New Stable Formulations of Recombinant Human Albumin-Human
Granulocyte Colony Stimulating Factor
[0001] The present application claims the benefit of U.S. Provisional Application No.
61/145,440 filed on January 16, 2009 and U.S. Provisional Application No. 61/145,436 filed
on January 16,2009. The contents of U.S. Provisional Application No. 61/145,440 and U.S.
Provisional Application No. 61/145,436 are hereby incorporated by reference in their
entirety.
BACKGROUND
[0002] Leukopenia is a reduction in the circulating White Blood Cells (WBC) and is often
defined as WBC count to < 4000/mL. The main cells involved in leukopenia are neutrophils.
However a reduced number of lymphocytes, monocytes, eosinophils, or basophils may also
contribute to the decreased total cell count (Merck Manual, 17th edition).
[0003] Neutropenia is characterized by a reduction in the blood neutrophil count, often
leading to increased susceptibility to bacterial and fungal infections. Neutropenia is
classified by the neutrophil count and the relative risk of infection: mild (1000 to 1500/mL),
moderate (grade 3, 500 to 1000/mL), or severe (grade 4, < 500/mL). Acute and severe
neutropenia is a life-threatening condition as it predisposes the patient to rapidly fatal
infections (Merck Manual, 17th edition).
[0004] Neutropenia can be caused by impaired production of neutrophils in the bone
marrow, or by accelerated destruction of neutrophils. Acute neutropenia may occur over a
few days when neutrophil use is rapid and production is severely impaired. Chronic
neutropenia may last for many months and is often caused by reduced production or
sequestration of neutrophils in the spleen. Neutropenia may be classified by whether it arises
secondary to factors extrinsic to marrow myeloid cells or whether an intrinsic defect appears
to be present in the myeloid progenitors (Merck Manual, 17th edition).
[0005] Neutropenia and its infectious complications are among the most common and
serious adverse effects of cytotoxic chemotherapy and other cancer therapies such as
radiation therapy, biotherapy, and bone marrow transplantation. Cytotoxic chemotherapy,
which works by seeking out and destroying fast-growing cells, induces neutropenia because

of the high proliferative rate of neutrophil precursors and the rapid turnover of blood
neutrophils (Merck Manual, 17th edition). The most common symptoms of neutropenia in
patients with undergoing chemotherapy include fever, mouth sores, and ear infections.
Patients with profound neutropenia often suffer from pyogenic infections such as septicemia,
cutaneous cellulitis, liver abscesses, furunculosis, pneumonia, stomatitis, gingivitis, perirectal
inflammation, colitis, sinusitis, and otitis media. Chemotherapy may have to be delayed until
the body can produce more neutrophils and a lower dosage may have to be given, resulting in
the treatment being less effective.
SUMMARY
[0006] The present invention is directed to compositions and methods useful in treating,
preventing or ameliorating diseases and conditions characterized by a lower than normal
white blood cell count, such as leukopenia and neutropenia. In some embodiments, the
compositions and methods include the recombinant human albumin-human granulocyte
colony stimulating factor shown in FIG. 9, or a variant or fragment thereof. In some
embodiments, the compositions and methods are used to treat, prevent or ameliorate
neutropenia and/or leukopenia, for example, neutropenia caused by the administration of
drugs, such as chemotherapy drugs administered for the treatment of cancer, can be treated
using the compositions of the invention.
[0007] In some embodiments, the compositions are pharmaceutical formulations and
include the recombinant human albumin-human granulocyte colony stimulating factor shown
in FIG. 9, or a fragment or variant thereof. In some embodiments, the pharmaceutical
formulation includes at least one pharmaceutically acceptable carrier, and has a pH of
between about 5 and about 8.0, between about 5 and about 7.5, between about 5 and about
7.2, between about 5 and about 7.0, between about 5 and about 6.8, between about 5 and
about 6.6, between about 5 and about 6.4, between about 5 and about 6.2, between about 5
and about 6, between about 6 and about 7.5, between about 6.0 and about 7.2, between about
6 and about 7. In other embodiments, the pH is about 4, about 4.2, about 4.4, about 4.5,
about 4.6, about 4.8 about 5, about 5.2 about 5.4 about 5.5, about 5.6, about 5.8, about 6.0,
about 6.2, about 6.4, about 6.5 about 6.6, about 6.8, about 7.0, about 7.2, about 7.4 about 7.5,
about 7.6, about 7.8 or about 8.0.

[0008] In some embodiments, the pharmaceutical composition includes recombinant human
albumin-human granulocyte colony stimulating factor at a concentration of between about 2.5
and about 240 mg/ml, between about 30 and about 120 mg/ml, between about 60 and about
120 mg/ml, about 5 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25
mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml,
about 55 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about
100 mg/ml, about 120 mg/ml, about 150 mg/ml, about 100 mg/ml, about 150 mg/ml, about
200 mg/ml, about 240 mg/ml, or about 250 mg/ml.
[0009] In some embodiments, the pharmaceutical composition includes at least one
pharmaceutically acceptable salt. In some embodiments, the salt is present in the
composition at a concentration of between about 5 and about 50 mM, between about 10 and
about 40 mM, between about 15 and about 30 mM, between about 20 and about 25 mM. In
some embodiments, the salt is present in the composition at a concentration of about 5 mM,
about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about
40 mM about 45 mM and about 50 mM.
[0010] In some embodiments, the pharmaceutical composition of includes at least one
pharmaceutically acceptable buffer. In some embodiments, the buffer is present in the
composition at a concentration of between about 5 and about 50 mM, between about 10 and
about 50 mM, between about 15 and about 50 mM, between about 5 and about 10 mM,
between about 10 and about 20 mM, between about 20 and about 30 mM, between about 15
and about 25 mM, or at about 20 mM. In some embodiments, the buffer is present in the
composition at a concentration of about 5 mM, about 10 mM, about 15 mM, about 20 mM,
about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about
55 mM or about 60 mM. In some embodiments, the buffer is a phosphate, a citrate, or a
combination thereof. In some embodiments, the buffer includes sodium phosphate, sodium
phosphate monobasic, sodium phosphate dibasic or a combination thereof.
[0011] In some embodiment, the pharmaceutical composition includes a feeze-drying
stabilizer. In some embodiments the freeze-drying stabilizer is trehalose dihydrate. In some
embodiments, the concentration of trehalose dihydrate is between about 20 to about 100 mM,
between about 40 to about 80 mM, between about 50 to about 70 mM, or about 60mM. In
some embodiments, the concentration of the stabilizer is about 20 mM, about 30 mM, about

40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100
mM, about 110 mM or about 120 mM.
[0012] In some embodiments, the pharmaceutical composition includes a bulking agent. In
some embodiments, the bulking agent is a poly-alcohol. In some embodiments, the poly-
alcohol is mannitol. In further embodiments, the pharmaceutical composition includes a
pharmaceutically acceptable carrier. In some embodiment, the carrier is polysorbate.
[0013] The pharmaceutical compositions described herein may be formulated for
administration in a number of forms. For example, in some embodiments, the pharmaceutical
compositions are prepared for oral, pulmonary, intravenous, intramuscular, subcutaneous,
rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal, intraperitoneal, ocular,
otic, local, buccal, nasal, or topical administration. Compositions may also be formulated for
specific dosage forms. For example, in some embodiments, the pharmaceutical composition
may be formulated as a liquid, gel, aerosol, ointment, cream, lyophilized formulation,
powder, cake, tablet, or capsule. In other embodiments, the pharmaceutical composition is
formulated as a controlled release formulation, fast melt formulation, delayed release
formulation, extended release formulation, pulsatile release formulation, and mixed
immediate release formulation. In some embodiments, the pharmaceutical composition is
provided as a liquid. In other embodiments, the pharmaceutical composition is provided as a
lyophilized powder. In still other embodiments, the pharmaceutical composition is provided
as a lyophilized cake.
[0014] The pharmaceutical composition described herein may be stored in a variety of
ways. In some embodiments, the pharmaceutical composition is stored in a vial; in other
embodiments, the pharmaceutical composition is stored in a syringe.
[0015] In some embodiments, the pharmaceutical composition comprises recombinant
human albumin-human granulocyte colony stimulating factor, at least one a buffer and/or pH
adjusting agent, and optionally at least one additional pharmaceutically acceptable carrier,
wherein the monomeric purity in solution of recombinant human albumin-human granulocyte
colony stimulating factor decreases by less than 10% after incubation at 25°C for 24 hours.
In some embodiments, the buffer is the same as the pH adjusting agent. In some
embodiments, the monomeric purity in solution after incubation at 25°C for 24 hours

decreases by less than about 1%, less than about 5%, less than about 15%, less than about
20% or less than about 25%.
[0016] In other embodiments, the pharmaceutical composition comprises recombinant
human albumin-human granulocyte colony stimulating factor, about 20 mM sodium
phosphate, about 180 mM mannitol, about 60 mM trehalose dihydrate, about 0.01% (w/v)
polysorbate 80, and pH of about 6.0, wherein the concentration of the recombinant human
albumin-human granulocyte colony stimulating factor is between about 2.5 mg/ml to about
120 mg/ml, or between about 30 mg/ml to about 60 mg/ml. In some embodiments, the
concentration of the recombinant human albumin-human granulocyte colony stimulating
factor is about 5, mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml,
about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about
55 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100
mg/ml, about 120 mg/ml, about 150 mg/ml, about 100 mg/ml, about 150 mg/ml, about 200
mg/ml, about 240 mg/ml, or about 250 mg/ml.
[0017] In still other embodiments, the pharmaceutical composition includes recombinant
human albumin-human granulocyte colony stimulating factor and PMTT20/6.0, wherein the
concentration of the recombinant human albumin-human granulocyte colony stimulating
factor is between about 2.5 and about 120 mg/ml, or between about 30 mg/ml to about 60
mg/ml. In some embodiments, the concentration of the recombinant human albumin-human
granulocyte colony stimulating factor is about 5, mg/ml, about 10 mg/ml, about 15 mg/ml,
about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about
45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80
mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 150 mg/ml, about 100
mg/ml, about 150 mg/ml, about 200 mg/ml, about 240 mg/ml, or about 250 mg/ml.
[0018] Both the foregoing general description and the following brief description of the
drawings and the detailed description are exemplary and explanatory and are intended to
provide further explanation of the invention as claimed. Other objects, advantages, and novel
features will be readily apparent to those skilled in the art from the following detailed
description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a graph showing that increasing rHSA-G-CSF concentration reduces
monomer purity. Monomer purity of rHSA-G-CSF samples at concentrations ranging from
2.5 to 240 mg/ml was measured by size exclusion high performance liquid chromatography
("SE-HPLC") after incubation in PMTT10/7.2 at 25°C for 24 hours.
[0020] FIG. 2 is a graph showing that increasing pH increases rHSA-G-CSF aggregation.
Aggregation of rHSA-G-CSF at a concentration of 15 mg/ml or 60 mg/ml was measured by
SE-HPLC after incubation at 25°C for 7 days in PMTT10 at a pH of 6.0, 6.8, 7.2, or 8.0.
[0021] FIG. 3 is a graph showing that increasing temperature increases rHSA-G-CSF
aggregation. Aggregation of rHSA-G-CSF at a concentration of 15 mg/ml or 60 mg/ml was
measured by SE-HPLC after incubation at 4°C, 25°C, or 40°C for 24 hours in PMTT 10/7.2.
[0022] FIG. 4 is a graph showing that increasing pH increases rHSA-G-CSF aggregation.
Aggregation of rHSA-G-CSF at a concentration of 48 mg/ml was measured by SE-HPLC
after incubation at 25°C for up to 3 days in PMTT10 at a pH of 5.8, 6.3, 6.4, or 7.0, or in
CMTT10 at a pH of 6.2. The top line is 10 mM PMTT pH 7.0; the line second from the top
is 10 mM PMTT pH 6.4; The third line from the top is 10 mm PMTT pH 6.3; the darker of
the lowest lines is 10 mM PMTT pH 5.8; the lighter of the lowest lines is 10 mM CMTT pH
6.2.
[0023] FIG. 5 is a graph showing that increasing salt concentration decreases rHSA-G-CSF
aggregation. Aggregation of rHSA-G-CSF at a concentration of 60 mg/ml was measured by
SE-HPLC after incubation for 1 day at 25°C/60% RH in PMTT10/7.2 and at a sodium
chloride concentration of 5 mM, 10 mM, 20 mM, or 50 mM.
[0024] FIG. 6 is a graph showing that increasing phosphate concentration decreases rHSA-
G-CSF aggregation. Aggregation of rHSA-G-CSF at a concentration of 60 mg/ml was
measured by SE-HPLC after incubation for 1 day at 25°C/60% relative humidity ("RH") in
PMTT/7.2 and at a phosphate concentration of 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or
50 mM.
[0025] FIG. 7 is a graph showing that purity of rHSA-G-CSF is maintained at
concentrations of up to 120 mg/ml in PMTT20/6.0 formulation buffer. Monomer purity of

rHSA-G-CSF samples at concentrations ranging from 2.5 to 120 mg/ml was measured by SE-
HPLC after incubation in PMTT10/7.2 (old buffer), or PMTT20/6.0 (new buffer) at 25°C for
24 hours.
[0026] FIG. 8 is a graph showing how pH and phosphate concentration effect aggregation
of rHSA-G-CSF. Aggregation of rHSA-G-CSF at a concentration of 60 mg/ml was measured
by SE-HPLC after incubation at 4°C or 25°C /60% RH for a maximal period of 14 days in
PMTT10/7.2 (old buffer) or in PMTT20/6.0 (new buffer).
[0027] FIG. 9A-C. FIG. 9A shows the nucleic acid and amino acid sequence of the rHSA-
G-CSF fusion polypeptide termed Neugranin™ ("NEUG"); FIG 9B shows the amino acid
sequence of human G-CSF; FIG. 9C shows the amino acid sequence of human serum
albumin.
[0028] FIG. 10 is a table showing aggregation measured by SE-HPLC of rHSA-G-CSF
incubated at different pHs, at a concentration of 48 mg/ml and at 25°C for up to 3 days.
[0029] FIG. 11 is a table showing aggregation of rHSA-G-CSF at different pH, protein
concentration and temperature measured by SE-HPLC. The top half of the table (first 3
entries) are at 15 mg/ml rHSA-G-CSF. The bottom half of the table (last three entries) are at
60 mg/ml rHSA-G-CSF.
[0030] FIG. 12 is a table showing the activity of rHSA-G-CSF after incubation at different
pHs, temperature and concentrations.
[0031] FIG. 13 is a flow chart showing an exemplary overview of manufacture of NEUG.
[0032] FIG. 14A and 14B show the results of SEC-HPLC and RP-HPLC, respectively,
comparing NEUG-1 ("IP") and NEUG-2 ("2P").
[0033] FIG. 15A and 15B show the comparability of charge heterogeneity by IEC-HPLC
and the comparability of identity by peptide mapping, respectively, of NEUG-1 ("IP") and
NEUG-2 ("2P").
[0034] FIG. 16 shows the comparable purity of NEUG-1 versus NEUG-2 on an SDS-
PAGE gel, stained with Coomassie blue.
[0035] FIG. 17A and 17B provide tables summarizing test results of three different lots of
NEUG-1 and five different lots of NEUG-2, prepared for clinical use in humans.

[0036] FIG. 18 shows Coomassie-stained SDS-PAGE analysis (reduced) of the Lot 2378-R
NEUG-1 reference standard.
[0037] FIG. 19A and 19B show chromatograms of SE-HPLC and RP-HPLC of Lot 2378-R
NEUG-1 reference standard.
[0038] FIG. 20 summarizes the results of analysis performed on a representative,
development lot of NEUG-2 final drug product.
[0039] FIG. 21 shows chromatograms of reverse phase ("RP"), ion-exchange ("IE") and
size exclusion ("SEC") chromatography of NEUG treated with hydrogen peroxide and TBO
or TBP; studies were performed to monitor oxidation of NEUG. NEUG control = medium
grey, NEUG treated with hydrogen peroxide = light grey; NEUG treated with TBP = black.
[0040] FIG. 22 is a table showing the results of NEUG treated with hydrogen peroxide and
TBO or TPB. Studies were performed to monitor the oxidation of NEUG.
[0041] FIG. 23 is a graph showing the median absolute neutrophil count (ANC) for
subjects receiving NEUG-1 before chemotherapy (Cycle 0) from treatment to day 14. At day
4, the lines from highest to lowest are: 300 µg/kg NEUG, 450 µg/kg NEUG 150 µg/kg and
50 µg/kg NEUG.
[0042] FIG. 24A and 24B are graphs showing the ANC and white blood cell ("WBC")
count for subjects who received NEUG-1 before and 24 hours after chemotherapy cycles.
NEUG dosages were as follows: 50 µg/kg; 150 µg/kg; 300 µg/kg; or 450µg/kg. The
neutrophil cut-offs for grade 3 and 4 neutropenia are shown by dashed lines in 24A; the
normal neutrophil range is also depicted in both 24A and 24B.
[0043] FIG. 25 is a graph showing the absolute neutrophil count (ANC) for subjects who
received 300 µg/kg NEUG, 450 µg/kg NEUG or 6 mg pegfilgrastim (Neulasta®)
approximately 24 hours following chemotherapy in cycle 1.
[0044] FIG. 26 illustrates the chemotherapy cycles for the Phase I studies.
[0045] FIG. 27 is a graph showing the pharmacokinetics of NEUG in the Phase I study in
human subjects. The serum concentration of NEUG administered subcutaneously at the
indicated doses (450 µg/kg, 300 µg/kg or 150 µg/kg) was measured in subjects with breast
cancer in the absence of chemotherapy. Squares: 450 µg/kg Cycle 0; triangles: 300 µg/kg

Cycle 0; circles: 150 µg/kg Cycle 0.
[0046] FIG. 28 is a graph showing the absolute neutrophil count ("ANC") for subjects in
Phase I. Subjects received 300 µg/kg NEUG (n=19), 450 µg/kg NEUG (n=20) or 6 mg
pegfilgrastim (Neulasta®) (n=9) in cycle 1 following study chemotherapy.
[0047] FIG. 29 is a graph showing the pharmacokinetics/pharmacodynamics ("PK/PD") of
NEUG in cycle 1 of chemotherapy (Phase I study). Patients received 450 µg/kg of NEUG
one day after doxorubicin/docetaxel administration in cycle 1. ANC is shown by the open
diamonds; NEUG concentration is shown by closed squares. Cut-offs for neutropenia grades
3 and 4 are shown by the dashed lines. The Lower Limit of Quantitation ("LLOQ") for
NEUG is shown as a dotted line at 6 ng/ml.
[0048] FIG. 30A and 30B show the area the curve (AUC) for each subject treated in Phase
I, Part B, based on the ANC values obtained for days 0 to 15. Figure 30A is a graph; the data
from Figure 23A is summarized in the table, 30B.
DETAILED DESCRIPTION
[0049] Disclosed herein are compositions and methods for treating, preventing and
ameliorating conditions and diseases characterized by a lowered white blood cell count. The
compositions and method described herein include a fusion polypeptide formed from human
serum albumin protein ("HSA") and human granulocyte-colony stimulating factor ("G-
CSF"). In one embodiment of the invention, the fusion polypeptide is 759 amino acids in
length; amino acids 1-585 of the fusion correspond to amino acids from the mature form of
HSA, and amino acids 586-759 of the fusion correspond to amino acids of the mature form of
human G-CSF. The amino acid sequences of the fusion protein is presented in FIG. 9A-9C.
[0050] Compositions and methods described herein also include therapeutic formulations
and pharmaceutical compositions comprising the recombinant HSA-G-CSF polypeptide. In
some embodiments, these formulations and compositions are configured for administration of
lower doses of the polypeptide, while in other embodiments, the formulations are configured
for administration of higher doses of the polypeptide.
[0051] The invention also encompasses fusion proteins comprising variants or fragments of
G-CSF, and fusion proteins comprising albumin or fragments or variants of albumin. The

invention also encompasses polynucleotides encoding the therapeutic albumin fusion proteins
of the invention, therapeutic albumin fusion proteins, compositions, pharmaceutical
compositions, formulations and kits. Host cells transformed with the polynucleotides
encoding therapeutic albumin fusion proteins are also encompassed by the invention, as are
methods of making the albumin fusion proteins of the invention using these polynucleotides,
and/or host cells.
[0052] In one embodiment, an albumin fusion protein according to the present invention
has extended shelf fife.
[0052] In a second embodiment, an albumin fusion protein according to the present
invention is more stable than the corresponding unfused G-CSF molecule.
[0053] The present invention further includes transgenic organisms modified to contain the
nucleic acid molecules of the invention, preferably modified to express an albumin fusion
protein of the invention.
[0054] The present invention relates generally to polynucleotides encoding albumin fusion
proteins; albumin fusion proteins; and methods of treating, preventing, or ameliorating
diseases or disorders using albumin fusion proteins or polynucleotides encoding albumin
fusion proteins. As used herein, "albumin fusion protein" refers to a protein formed by the
fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one
molecule of G-CSF (or fragment or variant thereof). An albumin fusion protein of the
invention comprises at least a fragment or variant of a G-CSF and at least a fragment or
variant of human serum albumin, which are associated with one another by genetic fusion
(i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a
polynucleotide encoding all or a portion of G-CSF is joined in-frame with a polynucleotide
encoding all or a portion of albumin). The G-CSF and albumin protein, once part of the
albumin fusion protein, may each be referred to as a "portion", "region" or "moiety" of the
albumin fusion protein (e.g., a "G-CSF protein portion" or an "albumin protein portion"). In a
highly preferred embodiment, an albumm fusion protein of the invention comprises at least
one molecule of G-CSF or fragment or variant of thereof (including, but not limited to a
mature form of the G-CSF protein) and at least one molecule of albumin or fragment or
variant thereof (including but not limited to a mature form of albumin).

[0055] In a further preferred embodiment, an albumin fusion protein of the invention is
processed by a host cell and secreted into the surrounding culture medium, Processing of the
nascent albumin fusion protein that occurs in the secretory pathways of the host used for
expression may include, but is not limited to signal peptide cleavage; formation of disulfide
bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and
O-linked glycosylation); specific proteolytic cleavages; and assembly into multimeric
proteins. An albumin fusion protein of the invention is preferably in the processed form. In a
most preferred embodiment, the "processed form of an albumin fusion protein" refers to an
albumin fusion protein product which has undergone N-terminal signal peptide cleavage,
herein also referred to as a "mature albumin fusion protein".
[0056] In one embodiment, the invention provides a polynucleotide encoding an albumin
fusion protein comprising, or alternatively consisting of, G-CSF and a serum albumin protein.
In a further embodiment, the invention provides an albumin fusion protein comprising, or
alternatively consisting of, G-CSF protein and a serum albumin protein. In other
embodiments, the invention provides an albumin fusion protein comprising, or alternatively
consisting of, a biologically active and/or therapeutically active fragment of G-CSF protein
and a serum albumin protein. In other embodiments, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a biologically active and/or
therapeutically active variant of G-CSF protein and a serum albumin protein. In preferred
embodiments, the serum albumin protein component of the albumin fusion protein is the
mature portion of serum albumin. The invention further encompasses polynucleotides
encoding these albumin fusion proteins.
[0057] In further embodiments, the invention provides an albumin fusion protein
comprising, or alternatively consisting of, G-CSF protein, and a biologically active and/or
therapeutically active fragment of serum albumin. In further embodiments, the invention
provides an albumin fusion protein comprising, or alternatively consisting of, G-CSF protein
and a biologically active and/or therapeutically active variant of serum albumin. In preferred
embodiments, the G-CSF protein portion of the albumin fusion protein is the mature portion
of the G-CSF protein. In a further preferred embodiment, the G-CSF protein portion of the
albumin fusion protein is the extracellular soluble domain of the G-CSF protein. In an
alternative embodiment, the G-CSF protein portion of the albumin fusion protein is the active

form of the G-CSF protein. The invention further encompasses polynucleotides encoding
these albumin fusion proteins.
[0058] In further embodiments, the invention provides an albumin fusion protein
comprising, or alternatively consisting of, a biologically active and/or therapeutically active
fragment or variant of G-CSF protein and a biologically active and/or therapeutically active
fragment or variant of serum albumin. In preferred embodiments, the invention provides an
albumin fusion protein comprising, or alternatively consisting of, the mature portion of G-
CSF protein and the mature portion of serum albumin. The invention further encompasses
polynucleotides encoding these albumin fusion proteins.
I. Definitions
[0053] The present invention is described herein using several definitions, as set forth
below and throughout the specification,
[0054] As used herein, "polynucleotide" refers to a nucleic acid molecule having a
nucleotide sequence encoding a fusion protein comprising, or alternatively consisting of, at
least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least
one molecule of Granulocyte-colony stimulating factor (G-CSF) (or fragment or variant
thereof)..
[0061] As used herein, "albumin fusion construct" refers to a nucleic acid molecule
comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of
albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide
encoding at least one molecule of G-CSF (or fragment or variant thereof); and, further
comprising, for example, one or more of the following elements: (1) a functional self-
replicating vector (including but not limited to, a shuttle vector, an expression vector, an
integration vector, and/or a replication system), (2) a region for initiation of transcription
(e.g., a promoter region, such as for example, a regulatable or inducible promoter, a
constitutive promoter), (3) a region for termination of transcription, (4) a leader sequence,
and (5) a selectable marker. The polynucleotide encoding the G-CSF and albumin protein,
once part of the albumin fusion construct, may each be referred to as a "poition," "region" or
"moiety" of the albumin fusion construct.
[0055] By a G-CSF polypeptide displaying a "therapeutic activity" or a G-CSF protein that
is "therapeutically active" is meant a G-CSF polypeptide that possesses one or more known

biological and/or therapeutic activities associated with G-CSF protein. As a non-limiting
example, a" G-CSF therapeutic protein" is a G-CSF protein that is useful to treat, prevent or
ameliorate a disease, condition or disorder. As a non-limiting example, a" G-CSF
therapeutic protein" may be one that binds specifically to a particular cell type (normal (e.g.,
lymphocytes) or abnormal e.g., (cancer cells)) and therefore may be used to target a
compound (drug, or cytotoxic agent) to that cell type specifically.
[0056] As used herein, the term "subject" is refers to an animal, preferably a mammal,
more preferably a human. The term "subject" and "patient" may be used interchangeably.
[0057] The term "pharmaceutically acceptable carrier" refers to any carrier that has
substantially no long term or permanent detrimental effect when administered to an
individual. Pharmaceutically acceptable carriers include diluents, fillers, salts, dispersion
media, coatings, emulsifying agents, wetting agents, sweetening or flavoring agents, tonicity
adjusters, absorption delaying agents, preservatives, antibacterial and antifungal agents,
buffers, pH adjusting agent, anti-oxidants, stabilizers, solubilizers, bulking agents,
cryoprotectant agents, aggregation inhibiting agents, or formulation auxiliary of any type.
Suitable carriers are described in Remington's Pharmaceutical Sciences (Remington's
Pharmaceutical Sciences, 2000,20th Ed., Lippincott, Williams & Wilkins), incorporated
herein by reference. Preferred examples of such carriers or diluents include, but are not
limited to, water, sodium chloride, mannitol, trehalose dehydrate, poiysorbate, such as
poiysorbate 80, various pharmaceutically acceptable buffers for adjusting pH (e.g. phosphate
buffers, citrate buffers, acetate buffers, and borate buffers).
[0058] The term "pharmaceutically acceptable salts" include those formed with anions such
as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those
formed with cations such as those derived from sodium, potassium, ammonium, calcium,
ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine,
etc.
[0059] The term "freeze-drying stabilizer" refers to a molecule that protects and reduces
chemical and/or physical instability of freeze-dried material. Preferred examples of freeze-
drying stabilizers include, but are not limited to, sucrose, trehalose, monosodium glutamate,
histidine, betaine, magnesium sulfate, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol,
mannitolpropyiene glycol, polyethylene glycol, pluronics, and combinations thereof. The

preferred freeze-drying stabilizer is a non-reducing sugar, such as trehalose dihydrate or
sucrose.
[0060] The term "bulking agent" refers to a compound which adds mass to the lyophilized
mixture and contributes to the physical structure of the lyophilized cake. Exemplary bulking
agents include sorbitol, glycine, mannitol, and polyethylene glycol.
[0061] The term "PMTT20/6.0" refers to a composition comprising sodium phosphate
monobasic (2.42 mg/mL, 17.4 mM), sodium phosphate dibasic (0.35 mg/mL, 2.5 mM),
mannitol (32.79 mg/mL, 180 mM), trehalose dehydrate (22.70 mg/mL, 60 mM), polysorbate
80 (0.1 mg/mL ,0.01%), the composition at a final pH of 6.0. This is also referred to a "new
buffer," "new PMTT" or "new formulation buffer," and is also described as comprising 20
mM phosphate, 180 mM mannitol, 60 mM trehalose dehydrate, 0.01% (w/v) polysorbate 80,
pH 6.0
[0062] The term "PMTT10/7.2" refers to a composition comprising 10 mM phosphate, 190
mM mannitol, 60 mM trehalose dihydrate, 0.01% (W/V) polysorbate 80, the composition at a
final pH of 7.2. This is also referred to as "old buffer," "old PMTT" or "old formulation
buffer."
[0063] The term "PMTT" refers to a composition comprising phosphate, mannitol,
trehalose dehydrate and polysorbate.
[0064] The term "CMTT10" refers to a composition comprising 10 mM sodium citrate, 190
mM mannitol, 60 mM trehalose dehydrate, 0.01% (W/V) polysporbate 80, buffer at pH of
6.2.
II. Granulocyte-colony stimulating factor
[0065] Granulocyte-colony stimulating factor (G-CSF) is a hematopoietic growth factor
that stimulates the production of neutrophils. Administration of G-CSF results in rapid
induction of a neutrophilic leukocytosis. Another important in vivo activity of G-CSF is
mobilization of hematopoietic progenitor cells into the peripheral blood (Duhrsen et al, 1988;
Molineux et al, 1999; Roberts et al, 1994). This effect includes not only the neutrophil
lineage but extends to other single lineage and multi-lineage progenitors and pluripotent
hematopoietic stem cells (Molineux et al, 1999). G-CSF also increases the cellular events
that are part of the defense mechanism against infections by priming neutrophils, thereby
increasing both their phagocytic and anti-bacterial activities against opsonized

Staphylococcus aureus. G-CSF also induces chemotaxis of neutrophils and monocytes and
adhesion of neutrophils (Yuo et al, 1989; Wang et al, 1988).
[0066] Recombinant G-CSF products are currently approved for a number of clinical
indications to stimulate the proliferation and differentiation of neutrophils. In clinical trials,
filgrastim (recombinant methionyl human G-CSF; Neupogen®, Amgen, Thousand Oaks,
CA) increased the number of peripheral neutrophils and thereby reduced the duration of
neutropenia after myelosuppressive chemotherapy. Filgrastim is given by daily subcutaneous
(SC) injection. Pegfilgrastim, a polyethylene glycol-conjugated rG-CSF, (Neulasta®), has
proven safe and effective as a once-per-cycle alternative to daily rG-CSF therapy to decrease
the incidence of febrile neutropenia in patients receiving myelosuppressive anti-cancer
drugs(Holmes, O'Shaughnessy et al, 2002; Green et al, 2003; Neulasta® SmPC 2007).
III. Human serum albumin
[0067] Human serum albumin (HSA) is the most prevalent naturally occurring blood
protein in the human circulatory system, measured at approximately 40 grams of
albumin/liter, persisting in the circulation for over 20 days. HSA lacks enzymatic or
immunological function and is widely distributed in vivo and is known to be a carrier for
therapeutic substances in the blood. Albumin is a carrier protein with minimal activity at
physiological concentrations. Both HSA and recombinant human albumin (rHSA) have the
same long circulating half-life in humans. Research has shown that therapeutic proteins
genetically fused to human albumin are able to take on the circulating half-life characteristics
of albumin (Syed et al, 1997). For example, a study in rabbits has shown that the half-life of
CD4 fused to albumin is 140 fold greater than non-fused CD4 (Yeh et al, 1992).
[0068] Human serum albumin, a protein of 585 amino acids in its mature form (as shown in
FIG. 1 of U.S. Patent No. 7,592,010, is responsible for a significant proportion of the osmotic
pressure of serum and also functions as a carrier of endogenous and exogenous ligands. At
present, HSA for clinical use is produced by extraction from human blood. The production
of recombinant HSA (rHSA) in microorganisms has been disclosed in EP 330 451 and EP
361 991.
W. Treatment with G-CSF

[0069] Primary prophylaxis with granulocyte colony-stimulating factors (G-CSF) is
recommended for the prevention of febrile neutropenia in patients who are at high risk based
on age, medical history, disease characteristics, and myelotoxicity of the chemotherapy
regimen. The American Society of Clinical Oncology (ASCO) and the European
Organization for Research and Treatment of Cancer (EORTC) recommends the use of G-CSF
when the risk of febrile neutropenia is approximately 20%. The U.S. National
Comprehensive Cancer Center Network (NCCN) recommends an optional indication of G-
CSF prophylaxis when the risk of febrile neutropenia is 10% to 20% and a definite indication
of G-CSF prophylaxis when the risk of febrile neutropenia is at least 20% (Smith et al, 2006,
Vogel et al 2005, Timmer-Bonte et al 2006, NCCN Guidelines).
[0070] Prophylaxis with colony-stimulating factors (CSFs) is recommended to alleviate the
toxicity of certain chemotherapy regimens. However, the added cost of these treatments is a
significant consideration both in the U.S. and especially in parts of the EU and may lead to
under-use of prophylactic G-CSF treatment and may also limit patient eligibility for dose-
intensive chemotherapy regimens (Timmer-Bonte et al, 2006; Adams et al, 2006, NCCN
Guidelines).
V. Polypeptide and Polynucleotide Fragments and Variants
A. Fragments
[0087] The present invention is further directed to fragments of G-CSF protein, albumin
proteins, and/or albumin fusion proteins of the invention. The present invention is also
directed to polynucleotides encoding fragments of the G-CSF protein, albumin proteins,
and/or albumin fusion proteins of the invention. Even if deletion of one or more amino acids
from the N-terminus of a protein results in modification or loss of one or more biological
functions of the G-CSF protein, albumin protein, and/or albumin fusion protein of the
invention, other therapeutic activities and/or functional activities (e.g., biological activities,
ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability
of polypeptides with N-terminal deletions to induce and/or bind to antibodies which
recognize the complete or mature forms of the polypeptides generally will be retained when
less than the majority of the residues of the complete polypeptide are removed from the N-
terminus. Whether a particular polypeptide lacking N-terminal residues of a complete
polypeptide retains such immunologic activities can readily be determined by routine

methods described herein and otherwise known in the art. It is not unlikely that a mutein
with a large number of deleted N-terminal amino acid residues may retain some biological or
immunogenic activities. In fact, peptides composed of as few as six amino acid residues may
often evoke an immune response.
[0071] Accordingly, fragments of G-CSF protein corresponding to a G-CSF protein portion
of an albumin fusion protein of the invention include the full length protein as well as
polypeptides having one or more residues deleted from the amino terminus of the amino acid
sequence of the reference polypeptide (i.e., a G-CSF protein, or a G-CSF protein portion of
an albumin fusion protein encoded by a polynucleotide or albumin fusion construct). In
particular, N-terminal deletions may be described by the general formula m to q, where q is a
whole integer representing the total number of amino acid residues in a reference polypeptide
(e.g., a G-CSF protein, or a G-CSF protein portion of an albumin fusion protein of the
invention), and m is defined as any integer ranging from 2 to q minus 6. Polynucleotides
encoding these polypeptides are also encompassed by the invention.
[0072] In addition, fragments of serum albumin polypeptides corresponding to an albumin
protein portion of an albumin fusion protein of the invention, include the full length protein
as well as polypeptides having one or more residues deleted from the amino terminus of the
amino acid sequence of the reference polypeptide (i.e., serum albumin, or a serum albumin
portion of an albumin fusion protein). In preferred embodiments, N-terminal deletions may
be described by the general formula m to 585, where 585 is a whole integer representing the
total number of amino acid residues in mature human serum albumin, and m is defined as any
integer ranging from 2 to 579. Polynucleotides encoding these polypeptides are also
encompassed by the invention. In additional embodiments, N-terminal deletions may be
described by the general formula m to 609, where 609 is a whole integer representing the
total number of amino acid residues in full length human serum albumin, and m is defined as
any integer ranging from 2 to 603. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0073] Moreover, fragments of albumin fusion proteins of the invention, include the full
length albumin fusion protein as well as polypeptides having one or more residues deleted
from the amino terminus of the albumin fusion protein. In particular, N-terminal deletions
may be described by the general formula m to q, where q is a whole integer representing the

total number of amino acid residues in the albumin fusion protein, and m is defined as any
integer ranging from 2 to q minus 6. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0074] Also as mentioned above, even if deletion of one or more amino acids from the N-
terminus or C-terminus of a reference polypeptide (e.g., a G-CSF protein; serum albumin
protein; or albumin fusion protein of the invention) results in modification or loss of one or
more biological functions of the protein, other functional activities (e.g., biological activities,
ability to multimerize, ability to bind a ligand) and/or therapeutic activities may still be
retained. For example, the ability of polypeptides with C-terminal deletions to induce and/or
bind to antibodies which recognize the complete or mature forms of the polypeptide generally
will be retained when less than the majority of the residues of the complete or mature
polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking the
N-terminal and/or C-terminal residues of a reference polypeptide retains therapeutic activity
can readily be determined by routine methods described herein and/or otherwise known in the
art.
[0075] The present invention further provides polypeptides having one or more residues
deleted from the carboxy terminus of the amino acid sequence of a G-CSF protein
corresponding to a G-CSF protein portion of an albumin fusion protein of the invention. In
particular, C-terminal deletions may be described by the general formula 1 to n, where n is
any whole integer ranging from 6 to q minus 1, and where q is a whole integer representing
the total number of amino acid residues in a reference polypeptide (e.g., a G-CSF protein, or
a G-CSF protein portion of an albumin fusion protein encoded by a polynucleotide or
albumin fusion construct). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0076] In addition, the present invention provides polypeptides having one or more residues
deleted from the carboxy terminus of the amino acid sequence of an albumin protein
corresponding to an albumin protein portion of an albumin fusion protein of the invention. In
particular, C-terminal deletions may be described by the general formula 1 to n, where n is
any whole integer ranging from 6 to 584, where 584 is the whole integer representing the
total number of amino acid residues in mature human serum albumin minus 1.
Polynucleotides encoding these polypeptides are also encompassed by the invention. In

particular, C-terminal deletions may be described by the general formula 1 to n, where n is
any whole integer ranging from 6 to 608, where 608 is the whole integer representing the
total number of amino acid residues in serum albumin minus 1. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0077] Moreover, the present invention provides polypeptides having one or more residues
deleted from the carboxy tenninus of an albumin fusion protein of the invention. In
particular, C-terminal deletions may be described by the general formula 1 to n, where n is
any whole integer ranging from 6 to q minus 1, and where q is a whole integer representing
the total number of amino acid residues in an albumin fusion protein of the invention.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[0078] In addition, any of the above described N- or C-terminal deletions can be combined
to produce a N- and C-terminal deleted reference polypeptide. The invention also provides
polypeptides having one or more amino acids deleted from both the amino and the carboxyl
termini, which may be described generally as having residues m to n of a reference
polypeptide (e.g., a G-CSF protein, or a G-CSF protein portion of an albumin fusion protein
of the invention, or serum albumin, or an albumin protein portion of an albumin fusion
protein of the invention, or an albumin fusion protein, or an albumin fusion protein encoded
by a polynucleotide or albumin fusion construct of the invention) where n and m are integers
as described above. Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0079] The present application is also directed to proteins containing polypeptides at least
about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98% or about 99% identical to a reference G-CSF
polypeptide or a reference albumin polypeptide set forth herein, or fragments thereof. In
preferred embodiments, the application is directed to proteins comprising polypeptides at
least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,
about 95%, about 96%, about 97%, about 98% or about 99% identical to reference
polypeptides having the amino acid sequence of N- and C-terminal deletions as described
above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
[0080] Preferred polypeptide fragments of the invention are fragments comprising, or
alternatively, consisting of, an amino acid sequence that displays a therapeutic activity and/or

functional activity (e.g. biological activity) of the polypeptide sequence of the G-CSF protein
or serum albumin protein of which the amino acid sequence is a fragment.
[0081] Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not necessarily
identical, to an activity of the polypeptide of the present invention. The biological activity of
the fragments may include an improved desired activity, or a decreased undesirable activity.
B. Variants
[0082J "Variant" refers to a polynucleotide or nucleic acid differing from a reference
nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are
overall closely similar, and, in many regions, identical to the reference nucleic acid or
polypeptide.
[0083] As used herein, "variant", refers to a G-CSF protein portion of an albumin fusion
protein of the invention, albumin portion of an albumin fusion protein of the invention, or
albumin fusion protein of the invention differing in sequence from a G-CSF protein, albumin
protein, and/or albumin fusion protein, respectively, but retaining at least one functional
and/or therapeutic property thereof as described elsewhere herein or otherwise known in the
art. Generally, variants are overall very similar, and, in many regions, identical to the amino
acid sequence of the G-CSF protein corresponding to a G-CSF protein portion of an albumin
fusion protein, albumin protein corresponding to an albumin protein portion of an albumin
fusion protein, and/or albumin fusion protein. Nucleic acids encoding these variants are also
encompassed by the invention.
[0084] The present invention is also directed to proteins which comprise, or alternatively
consist of, an amino acid sequence which is at least about 80%, about 85%, about 90%, about
91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99% or about 100%, identical to, for example, the amino acid sequence of a G-CSF
protein corresponding to a G-CSF protein portion of an albumin fusion protein of the
invention, albumin proteins corresponding to an albumin protein portion of an albumin
fusion protein of the invention, and/or albumin fusion proteins. Fragments of these
polypeptides are also provided. Further polypeptides encompassed by the invention are
polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic
acid molecule encoding an albumin fusion protein of the invention under stringent

hybridization conditions (e.g., hybridization to filter bound DNA in 6x. Sodium
chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes
in 0.2 x SSC, 0.1% SDS at about 50-65 degrees Celsius), under highly stringent conditions
(e.g., hybridization to filter bound DNA in 6 x sodium chloride/Sodium citrate (SSC) at about
45 degrees Celsius, followed by one or more washes in 0.1 x SSC, 0.2% SDS at about 68
degrees Celsius), or under other stringent hybridization conditions which are known to those
of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989 Current protocol in
Molecular Biology, Green publishing associates, Inc., and John Wiley & Sons Inc., New
York, at pages 6.3.1-6.3.6 and 2.10.3). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0085] By a polypeptide having an amino acid sequence at least, for example, 95%-
"identical" to a query amino acid sequence, it is intended that the amino acid sequence of the
subject polypeptide is identical to the query sequence except that the subject polypeptide
sequence may include up to five amino acid alterations per each 100 amino acids of the query
amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence
at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in
the subject sequence may be inserted, deleted, or substituted with another amino acid. These
alterations of the reference sequence may occur at the amino- or carboxy-terminal positions
of the reference amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference sequence or in one or more
contiguous groups within the reference sequence.
[0086] As a practical matter, whether any particular polypeptide is at least about 80%,
about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
96%, about 97%, about 98% or about 99% identical to, for instance, the amino acid sequence
of an albumin fusion protein of the invention or a fragment thereof (such as a G-CSF protein
portion of the albumin fusion protein or an albumin portion of the albumin fusion protein),
can be determined conventionally using known computer programs, A preferred method for
determining the best overall match between a query sequence (a sequence of the present
invention) and a subject sequence, also referred to as a global sequence alignment, can be
determined using the FASTDB computer program based on the algorithm of Brutlag et al.
(Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject

sequences are either both nucleotide sequences or both amino acid sequences. The result of
the global sequence alignment is expressed as percent identity. Preferred parameters used in
a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l,
Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window
Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the
length of the subject amino acid sequence, whichever is shorter.
[0087} If the subject sequence is shorter than the query sequence due to N- or C-terminal
deletions, not because of internal deletions, a manual correction must be made to the results.
This is because the FASTDB program does not account for N- and C-terminal truncations of
the subject sequence when calculating global percent identity. For subject sequences
truncated at the N- and C-termini, relative to the query sequence, the percent identity is
corrected by calculating the number of residues of the query sequence that are N- and C-
terminal of the subject sequence, which are not matched/aligned with a corresponding subject
residue, as a percent of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence alignment. This
percentage is then subtracted from the percent identity, calculated by the above FASTDB
program using the specified parameters, to arrive at a final percent identity score. This final
percent identity score is what is used for the purposes of the present invention. Only residues
to the N- and C-termini of the subject sequence, which are not matched/aligned with the
query sequence, are considered for the purposes of manually adjusting the percent identity
score. That is, only query residue positions outside the farthest N- and C-terminal residues of
the subject sequence.
[0088] For example, a 90 amino acid residue subject sequence is aligned with a 100 residue
query sequence to determine percent identity. The deletion occurs at the N-terminus of the
subject sequence and therefore, the FASTDB alignment does not show a matching/alignment
of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the
sequence (number of residues at the N- and C-termini not matched/total number of residues
in the query sequence) so 10% is subtracted from the percent identity score calculated by the
FASTDB program. If the remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject sequence is compared with
a 100 residue query sequence. This time the deletions are internal deletions so there are no

residues at the N- or C-termini of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by FASTDB is not manually corrected.
Once again, only residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the
query sequence are manually corrected for. No other manual corrections are to made for the
purposes of the present invention.
[0089] The variant will usually have at least about 75% (in other embodiments at least
about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98% or about 99%) sequence identity with a length of
normal HA or G-CSF protein which is the same length as the variant. Homology or identity
at the nucleotide or amino acid sequence level is determined by BLAST (Basic Local
Alignment Search Tool) analysis using the algorithm employed by the programs blastp,
blastn, blastx, tblastn and tblastx (Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264-2268
(1990) and Altschul, J. Mol. Evol. 36: 290-300 (1993), fully incorporated by reference)
which are tailored for sequence similarity searching.
[0090] The approach used by the BLAST program is to first consider similar segments
between a query sequence and a database sequence, then to evaluate the statistical
significance of all matches that are identified and finally to summarize only those matches
which satisfy a preselected threshold of significance. For a discussion of basic issues in
similarity searching of sequence databases, see Altschul et al., (Nature Genetics 6:119-129
(1994)) which is fully incorporated by reference. The search parameters for histogram,
descriptions, alignments, expect (i.e., the statistical significance threshold for reporting
matches against database sequences), cutoff, matrix and filter are at the default settings. The
default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix
(Henikoff et al, Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992), fully incorporated by
reference). For blastn, the scoring matrix is set by the ratios of M (i.e., the reward score for a
pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the
default values for M and N are 5 and -4, respectively. Four blastn parameters may be adjusted
as follows: Q-10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates
word hits at every wink.sup.th position along the query); and gapw=16 (sets the window
width within which gapped alignments are generated). The equivalent Blastp parameter

settings were Q=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences,
available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation
penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein
comparisons are GAP=8 and LEN=2.
[0091] The polynucleotide variants of the invention may contain alterations in the coding
regions, non-coding regions, or both. Especially preferred are polynucleotide variants
containing alterations which produce silent substitutions, additions, or deletions, but do not
alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by
silent substitutions due to the degeneracy of the genetic code are preferred. Moreover,
polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than
10, or 5-50,5-25, 5-10,1-5, or 1-2 amino acids are substituted, deleted, or added in any
combination are also preferred. Polynucleotide variants can be produced for a variety of
reasons, e.g., to optimize codon expression for a particular host (change codons in the human
mRNA to those preferred by a bacterial host, such as, yeast or E. coli).
[0092] In a preferred embodiment, a polynucleotide of the invention which encodes the
albumin portion of an albumin fusion protein is optimized for expression in yeast or
mammalian cells. In a further preferred embodiment, a polynucleotide of the invention
which encodes the G-CSF protein portion of an albumin fusion protein is optimized for
expression in yeast or mammalian cells. In a still further preferred embodiment, a
polynucleotide encoding an albumin fusion protein of the invention is optimized for
expression in yeast or mammalian cells.
[0093] In an alternative embodiment, a codon optimized polynucleotide which encodes a
G-CSF protein portion of an albumin fusion protein does not hybridize to the wild type
polynucleotide encoding the G-CSF protein under stringent hybridization conditions as
described herein. In a further embodiment, a codon optimized polynucleotide which encodes
an albumin portion of an albumin fusion protein does not hybridize to the wild type
polynucleotide encoding the albumin protein under stringent hybridization conditions as
described herein. In another embodiment, a codon optimized polynucleotide which encodes
an albumin fusion protein does not hybridize to the wild type polynucleotide encoding the G-
CSF protein portion or the albumin protein portion under stringent hybridization conditions
as described herein.

[0094] In an additional embodiment, a polynucleotide which encodes a G-CSF protein
portion of an albumin fusion protein does not comprise, or alternatively consist of, the
naturally occurring sequence of that G-CSF protein. In a further embodiment, a
polynucleotide which encodes an albumin protein portion of an albumin fusion protein does
not comprise, or alternatively consist of, the naturally occurring sequence of albumin protein.
In an alternative embodiment, a polynucleotide which encodes an albumin fusion protein
does not comprise, or alternatively consist of, the naturally occurring sequence of a G-CSF
protein portion or the albumin protein portion.
[0095] Using known methods of protein engineering and recombinant DNA technology,
variants may be generated to improve or alter the characteristics of the polypeptides of the
present invention. For instance, one or more amino acids can be deleted from the N-terminus
or C-terminus of the polypeptide of the present invention without substantial loss of
biological function.
[0096] In preferred embodiments, the variants of the invention have conservative
substitutions. By "conservative substitutions" is intended swaps within groups such as
replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement
of the hydroxyl residues Ser and Tar; replacement of the acidic residues Asp and Glu;
replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg,
and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the
small-sized amino acids Ala, Ser, Thr, Met, and Gly.
[0097] Guidance concerning how to make phenotypically silent amino acid substitutions is
provided, for example, in Bowie et al, "Deciphering the Message in Protein Sequences:
Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors
indicate that there are two main strategies for studying the tolerance of an amino acid
sequence to change.
[0098] The first strategy exploits the tolerance of amino acid substitutions by natural
selection during the process of evolution. By comparing amino acid sequences in different
species, conserved amino acids can be identified. These conserved amino acids are likely
important for protein function. In contrast, the amino acid positions where substitutions have
been tolerated by natural selection indicates that these positions are not critical for protein

function. Thus, positions tolerating amino acid substitution could be modified while still
maintaining biological activity of the protein.
[0099] The second strategy uses genetic engineering to introduce amino acid changes at
specific positions of a cloned gene to identify regions critical for protein function. For
example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single
alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells,
Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for
biological activity.
[0100] As the authors state, these two strategies have revealed that proteins are surprisingly
tolerant of amino acid substitutions. The authors further indicate which amino acid changes
are likely to be permissive at certain amino acid positions in the protein. For example, most
buried (within the tertiary structure of the protein) amino acid residues require nonpolar side
chains, whereas few features of surface side chains are generally conserved. Moreover,
tolerated conservative amino acid substitutions involve replacement of the aliphatic or
hydrophobic amino acids Ala, Val, Leu and Be; replacement of the hydroxyl residues Ser and
Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn
and Gin, replacement of the basic residues Lys. Arg, and His; replacement of the aromatic
residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr,
Met, and Gly. Besides conservative amino acid substitution, variants of the present invention
include (i) polypeptides containing substitutions of one or more of the non-conserved amino
acid residues, where the substituted amino acid residues may or may not be one encoded by
the genetic code, or (ii) polypeptides containing substitutions of one or more of the amino
acid residues having a substituent group, or (iii) polypeptides which have been fused with or
chemically conjugated to another compound, such as a compound to increase the stability
and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) polypeptide
containing additional amino acids, such as, for example, an IgG Fc fusion region peptide.
Such variant polypeptides are deemed to be within the scope of those skilled in the art from
the teachings herein.
[0101] For example, polypeptide variants containing amino acid substitutions of charged
amino acids with other charged or neutral amino acids may produce proteins with improved
characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both

reduces activity and increases clearance due to the aggregate's immunogenic activity. See
Pinckard et at, Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al, Diabetes 36: 838-845
(1987); Cleland et al., Crit Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
[0102] In specific embodiments, the polypeptides of the invention comprise, or
alternatively, consist of, fragments or variants of the amino acid sequence of an albumin
fusion protein, me amino acid sequence of a G-CSF protein and/or human serum albumin,
wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50,10-50 or 50-150, amino acid
residue additions, substitutions, and/or deletions when compared to the reference amino acid
sequence. In preferred embodiments, the amino acid substitutions are conservative. Nucleic
acids encoding these polypeptides are also encompassed by the invention.
[0103] The polypeptide of the present invention can be composed of amino acids joined to
each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may
contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be
modified by either natural processes, such as post-translational processing, or by chemical
modification techniques which are well known in the art. Such modifications are well
described in basic texts and in more detailed monographs, as well as in a voluminous research
literature. Modifications can occur anywhere in a polypeptide, including the peptide
backbone, the amino acid side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in the same or varying degrees
at several sites in a given polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and
they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic
polypeptides may result from posttranslation natural processes or may be made by synthetic
methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation,
covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of
a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links, formation of cysteine, formation
of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic
processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-

RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
(See, for instance, PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-
TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,
Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646
(1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:4862 (1992)).
C. Functional Activity
[0104] "A polypeptide having functional activity" refers to a polypeptide capable of
displaying one or more known functional activities associated with the full-length, pro-
protein, and/or mature form of a G-CSF protein. Such functional activities include, but are
not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide
for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody
which binds to a specific polypeptide of the invention), ability to form multimers with
polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
[0105] "A polypeptide having biological activity" refers to a polypeptide exhibiting activity
similar to, but not necessarily identical to, an activity of a G-CSF protein of the present
invention, including mature forms, as measured in a particular biological assay, with or
without dose dependency.
[0106] In preferred embodiments, an albumin fusion protein of the invention has at least
one biological and/or therapeutic activity associated with the G-CSF protein portion (or
fragment or variant thereof) when it is not fused to albumin.
[0107] The albumin fusion proteins of the invention can be assayed for functional activity
(e.g., biological activity) using or routinely modifying assays known in the art, as well as
assays described herein. Additionally, one of skill in the art may routinely assay fragments of
a G-CSF protein corresponding to a G-CSF protein portion of an albumin fusion protein.
Further, one of skill in the art may routinely assay fragments of an albumin protein
corresponding to an albumin protein portion of an albumin fusion protein, for activity using
assays known in the art and/or as described in the Examples section below.
[0108] For example, in one embodiment where one is assaying for the ability of an albumin
fusion protein to bind or compete with a G-CSF protein for binding to an anti- G-CSF

polypeptide antibody and/or anti-albumin antibody, various immunoassays known in the art
can be used, including but not limited to, competitive and non-competitive assay systems
using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope
labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation assays,
immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In
one embodiment, antibody binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by detecting binding of a secondary
antibody or reagent to the primary antibody. In a further embodiment, the secondary
antibody is labeled. Many means are known in the art for detecting binding in an
immunoassay and are within the scope of the present invention.
[0109] In a preferred embodiment, where a binding partner (e.g., a receptor or a ligand) of a
G-CSF protein is identified, binding to that binding partner by an albumin fusion protein
which comprises that G-CSF protein as the G-CSF protein portion of the fusion can be
assayed, e.g., by means well-known in the art, such as, for example, reducing and non-
reducing gel chromatography, protein affinity chromatography, and affinity blotting. See
generally, Phizicky et at, Microbiol. Rev. 59:94-123 (1995). In another embodiment, the
ability of physiological correlates of an albumin fusion protein to bind to a recepto(s) of the
G-CSF polypeptide corresponding to the G-CSF protein portion of the fusion can be routinely
assayed using techniques known in the art.
[0110] In an alternative embodiment, where the ability of an albumin fusion protein to
multirnerize is being evaluated, association with other components of the multimer can be
assayed, e.g., by means well-known in the art, such as, for example, reducing and non-
reducing gel chromatography, protein affinity chromatography, and affinity blotting. See
generally, Phizicky et al, supra.
[0111] Immunoassays which can be used to analyze binding and cross-reactivity and to
confirm the identity of a HAS-G-CSF fusion include, but are not limited to, competitive and
non-competitive assay systems using techniques such as western blots, radioimmunoassays,
ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays,

immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to
name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al,
eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
York, which is incorporated by reference herein in its entirety).
[0112] Antibodies that bind a G-CSF protein corresponding to the G-CSF protein portion of
an albumin fusion protein may also be described or specified in terms of their binding affinity
for a given protein or antigen, preferably the antigen which they specifically bind. Preferred
binding affinities include those with a dissociation constant or Kd less than 5 x 10-2 M, 10-2
M, 5 x 10-3 M, 10-3 M, 5 x 10-4 M, 10-4 M. More preferred binding affinities include those
with a dissociation constant or Kd less than 5 x 10-5 M, 10-5 M, 5 x 10-6 M, 10-6 M, 5 x 10-7
M, 10-7 M, 5 x 10-8 M or 10-8 M. Even more preferred binding affinities include those with a
dissociation constant or Kd less than 5 x 10-9 M, 10-9 M, 5 x 10-10 M, 10-10 M, 5 x 10-11 M, 10-
11 M, 5 x 10-12 M, 10-12 M, 5 x 10-13 M, 10-13 M, 5 x 10-14 M, 10-14 M, 5 x 10-15 M, or 10-15 M.
In addition, assays described herein and otherwise known in the art may routinely be applied
to measure the ability of albumin fusion proteins and fragments, variants and derivatives
thereof to elicit biological activity and/or G-CSF activity (either in vitro or in vivo) related to
either the G-CSF protein portion and/or albumin portion of the albumin fusion protein. Other
methods will be known to the skilled artisan and are within the scope of the invention.
VI. BSA-G-CSF fasten protein
[0113] Recombinant human albumin-human granulocyte colony stimulating factor (rHSA-
G-CSF) is a G-CSF analogue. Examples of rHSA-G-CSFs are described in U.S. Patent No.
5,665,863 and in U.S. Patent No. 7,041,478 hereby incorporated by reference.
[0114] Another example of rHSA-G-CSF is Neugranin™ which is being developed by
Teva Biopharmaceuticals USA LTD. Neugranin™ ("NEUG") is a 759 amino acid fusion
polypeptide with a molecular mass of approximately 85 kDa connected in a single chain.
Residues 1-585 correspond to the mature form of HSA, and residues 586-759 correspond to
the mature form of human G-CSF. The NeugraninTM fusion polypeptide is shown in FIG. 9.
VH. Producing the fusion protein

[0115] Examples of the synthetic process of manufacture of rHSA-G-CSF are described in
U.S. Patent Application Serial No. 11/929,828 hereby incorporated by reference in its
entirety. In some embodiments, NEUG is produced using a yeast host system
(Saccharomyces cerevisiae) genetically engineered to express the NEUG fusion protein.
NEUG is harvested from the fermentation medium of the yeast culture and purified using
methods well known in the art (e.g., by a series of chromatography and filtration steps, such
as affinity chromatography and ion exchange chromatography).
[0116] In one non-limiting example, a NEUG fusion construct was developed as follows.
The full-length albumin cDNA was isolated from a human cDN A library in the laboratory of
Dr. F.E. Baralle at the University of Oxford, UK. This clone was sent to Delta
Biotechnology Limited, Nottingham, UK, as the plasmid pAT153ALB. In addition, the 6-
amino acid HS A pro-peptide (RGVFRR) was modified to facilitate more efficient processing
in yeast (RSLDKR).
[0117] The Neugranin production plasmid is based on the 2-u plasmid found in wild type
Saccharomyces cerevisiae. The pSAC35-based expression vector (patents EP 286 424 B, US
5,637,504) contains the LEU2 gene from Saccharomyces cerevisiae as a selectable marker
that complements the leucine-deficiency of the production host. This production plasmid
also contains a strong yeast promoter, PRB1, and sequences from plasmid pUC9 that permit
cloning and propagation in E. coli. In addition, the plasmid exhibits the unique attribute in
which it eliminates the pUC9-derived sequences required for propagation in E. coli once
transformed into yeast. This is accomplished by flanking FLP recognition targets and the
expression of the yeast FLP recombinase from the plasmid once in yeast. Thus, no bacterial
DNA is present in the organism used for production of NEUG. This is confirmed by rescue
and sequence of the 2um plasmid from the yeast after the master cell bank is generated.
[0118] As described above, the NEUG production plasmid, termed CID1643
(pSAC35:HSA.GCSF(T31-P204)), was derived from the pSAC35-based expression vector.
The region corresponding to T31-P204 of human G-CSF was amplified by PCR, while
adding the appropriate 5' and 3' restriction sites to permit a seamless fusion to the 3'-end of
the HSA open reading frame.
[0119] NEUG seed vials were used to prepare a cGMP master cell bank (MCB) at Human
Genome Sciences, Inc., (HGS) in Rockville, MD. The testing and characterization of the
-

NEUG MCB was undertaken at Charles River Laboratories (Malvern, PA, USA) and Lark
Technologies (Houston, TX, USA) in compliance with the ICH guideline Q5D (Derivation
and Characterization of Cell Substrates Used for Production of Biotechnological/Biologicals
Products).
[0120] A cGMP working cell bank (WCB) derived from this MCB was subsequently
generated and tested at Charles River Laboratories (Malvern, PA, USA).
[0121] All media components used in the manufacture of the NEUG cell line banks were
synthetic, biosynthetic or plant derived. No components of animal or human origin were
used during cell line or cell bank preparation.
[0122] The cell banks is stored at < -135°C in a cryopreservation media in pre-sterilized 1.8
mL Nunc polypropylene tubes with internally threaded caps.
[0123] Non-limiting, exemplary methods of isolating, purifying and preparing the rHSA-G-
CSF fusion protein for pharmaceutical use are provided below, in the Experimental
Examples.
VIII. Preparation of therapeutic proteins
[0124] Therapeutic proteins in their native state or when recombinantly produced, such as
interferons and growth hormones, are typically labile molecules exhibiting short shelf-lives,
particularly when formulated in aqueous solutions. The instability in these molecules when
formulated for administration dictates that many of the molecules must be lyophilized and
refrigerated at all times during storage, thereby rendering the molecules difficult to transport
and/or store. Storage problems are particularly acute when pharmaceutical formulations must
be stored and dispensed outside of the hospital environment. Albumin fusion proteins
comprising a therapeutic protein have demonstrated extended shelf life compared to the shelf
life of the same therapeutic protein when not fused to albumin. Shelf-life typically refers to
the time period over which the therapeutic activity of a therapeutic protein in solution or in
some other storage formulation, is stable without undue loss of therapeutic activity.
[0125] Nevertheless, aggregation and chemical instability are major obstacles in developing
formulations for albumin fusion proteins comprising a therapeutic protein. Aggregation of
pharmaceutical formulations can be very deleterious since it reduces activity, and increases

drug clearance due to the aggregate's immunogenic activity (Pinckard et al. 1967; Robbins et
al. 1987; ClelandetaL, 1993).
[0126] Presented below are a number of non-limiting examples which illustrate various
aspects of the methods and compositions described herein.
IX, Experimental Examples
[0127] The following examples are given to illustrate the present invention. It should be
understood, however, that the invention is not to be limited to the specific conditions or
details described in these examples. All publically available documents referenced herein,
including but not limited to US patents, are hereby incorporated by reference.
[0128] In Examples 1, 2, and 6, three different, non-limiting formulations of
pharmaceutical Neugranin™ ("NEUG") compositions are presented. Each formulation
comprises the NEUG fusion polypeptide shown in FIG. 9. The method and resulting
composition described in section A are broadly referred to as "NEUG-0." The method and
resulting composition described in section B are broadly referred to as "NEUG-1," and the
method and resulting composition described in section E are broadly referred to as "NEUG-
2."
[0129] NEUG is a fusion polypeptide with a molecular mass of approximately 85 kDa
connected in a single chain comprising residues 1-585 corresponding to the mature form of
HSA, and residues 586-759 corresponding to the mature form of human G-CSF. The amino
acid sequence of NEUG fusion protein is shown in FIG. 9.
Example 1: Preparation of therapeutic NEUG-0
[0130] NEUG-0 was manufactured for use in toxicology experiments. The formulation
included NEUG at 4.0 mg/ml in 10 mM citrate, 75 mM sodium chloride, 100 mM sucrose,
0.01% polysorbate 80, and was buffered to pH 6.5,
Safety/Toxicology
[0131] The non-clinical safety of NEUG was assessed in monkeys. Repeat exposure (once-
weekly for 4 weeks) at 100,500,1000 µg/kg NEUG was well tolerated, with no mortality,
morbidity, or changes in body weight or food consumption. There were not gross or

microscopic changes that were considered adverse toxicities. Treatment related observations
were limited to expected pharmacology of G-CSF administration (e.g., neutrophilia, slight
increase in spleen weight, myeloid hyperplasia) and were not considered toxicities. The
NOAEL in monkey was > 1 mg/kg/week, which was ~12 fold higher than observed
following a dose of 0.45 mg/kg in humans. Treated animals also showed an increase in ANC
and WBC. (Data not shown).
Example 2: Preparation of therapeutic NEUG-1
[0132] NEUG-1 was manufactured for clinical studies in humans. The formulation
included NEUG at 15.0 mg/ml, in PMMT10/7.2 (10 mM sodium phosphate, 200 mM
mannitol, 60 mm trehalose dehydrate, 0.01% (W/V) polysorbate-80, at pH 7.2.
[0133] The composition of NEUG-1 is provided in Table 1, below.

aA 0.16 mL overage is included in each vial to assure the 1.0 mL deliverable volume.
[0134] One exemplary method for the manufacture of bulk drug substance ("BDS") for
NEUG-1 is shown by the non-limiting process steps of FIG. 13. The BDS is then stored at
about -80°C (nominal value, acceptable range of storage temperature is about -65°C).
[0135] To improve the robustness of the formulation for shipping and storage at clinical
sites, as well as to provide a stable product with an expected long shelf life, a lyophilized
form of NEUG-1 has been developed. Methods of freeze-drying are well known in the art,

and one exemplary method, which forms elegant, lyophilized cakes, was performed as
described in the following steps.
Step 1: thawing and dilution of bulk drug substance
[0136] Containers of NEUG bulk drug substance are thawed at 15 to 30°C. The bulk drug
substance is pooled (if necessary), mixed, and protein concentration determined by
absorbance at 280 ran. The bulk drug substance concentration is used to calculate the amount
of dilution buffer required. Density and pH are also measured. Formulation buffer is used to
dilute the bulk drug substance to the desired final concentration.
Step 2; sterile filtration
[0137] The diluted solution is transferred from the formulation area into a sterile receiving
vessel through sterile filters that have been integrity tested. The filtration-train consists of two
0.2 µm PVDF filters in series. All filters are previously unused and are integrity tested and
disposed of post-manufacturing.
Step 3: filling and partial stoppering
[0138] Neugranin is filled into depyrogenated 3 mL USP Type I glass vials, partially
stoppered, and trayed onto bottomless lyophilization trays. Weight checks are performed
throughout the filling operation to verify fill volume.
Step 4: lyophilization
[0139] The lyophilization cycle is controlled by chamber pressure and shelf temperature.
Product temperature is monitored by product thermocouples placed throughout the
lyophilization chamber. Partially stoppered vials are loaded onto pre-chilled shelves and
frozen at a controlled rate. An annealing step is performed to promote crystallization of
mannitol (bulking agent). Primary drying is then performed and verified by monitoring
thermocouples. Secondary drying is performed to achieve the desired moisture content. At
the end of secondary drying, the vacuum in the lyophilization chamber is released by venting
0.2 µm sterile-filtered nitrogen into the chamber.
Step 5; sealing and capping

[0140] The stoppers are seated into the vials under a partial vacuum (ca 11-12 psia). The
product trays are unloaded and the vials are capped.
[0141] The initial lyophilized clinical material (NEUG-1) has proven quite stable, with a
current shelf-life of 2 years (when stored at 2-8°C) which will likely be further extended as
data from continuing stability studies is reviewed.
Example 3: General properties of a NEUG-1 reference standard, Lot 2378-R
[0142] A variety of methods have been developed to examine the physiochemical and
biological characteristics of NEUG. One lot of NEUG termed reference standard Lot 2378-
R, contains 21.3 mg/mL NEUG and was formulated in 10 mM sodium phosphate, 200mM
mannitol, 60mM trehalose dehydrate, and 0.01% (w/v) polysorbate 80 at pH 7.2
(PMTT10/7.2). Physicochemical characterization was performed on the reference standard
lot and the test attributes, analytical methods and a summary of results is provided in Table 2,
below.



[0143] SDS-PAGE analysis of NEUG-1 Lot 2378-R with Coomassie blue stain results in a
single band (FIG. 19). A representative SEC-HPLC and RP-HPLC chromatogram of
NEUG-1 Lot 2378-R is also shown in FIGS. 19A and 19B, respectively.
Example 4: Testing NEUG-0 and NEUG-1 Formulations
1, Effect of Protein Concentration on rHSA-G-CSF Monomer Purity
[0144] Recombinant human albumin-human granulocyte colony stimulating factor (rHSA-
G-CSF) at concentrations ranging from 2.5 to 240 mg/ml was incubated in PMTT 10/7.2
(10mM Phosphate, 190 mM mannitol, 60 mM trehalose dihydrate, 0.01% (W/V) polysorbate
80, pH 7.2) at 25°C for 24 hours. Subsequent to incubation, monomer purity was measured
by SE-HPLC.
[0145] The results (FIG. 1) show that aggregation increased with increasing rHSA-G-CSF
concentration,
2. Effect of pH on rHSA-G-CSF Aggregation
[0146] Recombinant human albumin-human granulocyte colony stimulating factor at a
concentration of 15 mg/ml and 60 mg/ml was incubated at 25°C for 7 days in PMTT 10
(10mM Phosphate, 190 mM Mannitol, 60 mM Trehalose Dihydrate, 0.01% (W/V)
Poiysorbate 80) at a pH of 6.0,6.8, 7.2, and 8,0. Subsequent to incubation rHA-G-CSF
monomer purity was measured by SE-HPLC.
[0147] The results (FIG. 2) demonstrate that aggregation increased with increasing pH, and
accelerated by increased protein concentration.

3. Effect of Temperature on rHSA-G-CSF Aggregation
[0148] Recombinant human albumin-human granulocyte colony stimulating factor at a
concentration of 60 mg/ml was incubated in PMTT10/7.2 buffer at 4°C, 25°C, or 40°C for 24
hours. Subsequent to incubation rHSA-G-CSF monomer purity was measured by SE-HPLC.
[0149] The results (FIG. 3) demonstrate that aggregation was accelerated with increasing
temperature.
4. Effect of pH on rHSA-G-CSF Aggregation
[0150] Recombinant human albumin-human granulocyte colony stimulating factor at a
concentration of 48 mg/ml was incubated at 25°C for up to 3 days in PMTT10 (10mM
phosphate, 190 mM mannitol, 60 mM trehalose dihydrate, 0.01% (W/V) polysorbate 80) at a
pH of 5.8,6.3,6.4, or 7.0, or in CMTT10 (lOmM sodium citrate, 190 mM mannitol, 60 mM
trehalose dihydrate, 0.01% (W/V) polysorbate 80) buffer at a pH of 6.2. Recombinant human
albumin-human granulocyte colony stimulating factor monomer purity was measured by SE-
HPLC at day 0,1,2, and 3 after incubation.
[0151] The results (FIG. 4, and FIG. 10) demonstrate that most of the aggregation occurred
in the first day of incubation and that the rate of aggregation increased due to increasing pH.
5. Effect of pH on rHSA-G-CSF Aggregation
[0152] Recombinant human albumin-human granulocyte colony stimulating factor at a
concentration of 15 mg/ml or 60 mg/ml was incubated at 4°C, 25°C, or 40°C for up to 10 days
in PMTT10 at a pH of 6.0, 6.8,7.2, or 8.0. Monomer purity was measured by SE-HPLC at
day 0,1, 7, and 10 after incubation.
[0153] The results (FIG. 11) demonstrate that aggregation increased with increasing pH,
and accelerated by increased temperature and protein concentration.
6. Effect of pH on rHSA-G-CSF Bioactivity
[0154] Recombinant human albumin-human granulocyte colony stimulating factor at a
concentration of 15 mg/ml or 60 mg/ml was incubated at 4°C, 25°C, or 40°C for up to 10 days
in PMTT10 at a pH of 6.0,6.8,7.2, or 8.0. Bioactivity was measured by Bioassay at day 0,
and either day 7 or day 10 after incubation.

[0155] Briefly, NFS-60 cells proliferate in response to both G-CSF and NEUG. In this
assay, 10,000 cell/well were plated in a 96 well microplate and were treated with NEUG
proteins for 20 hours at 37°C. After a 20-hour incubation the NFS-60 cells were pulsed with
0.5 µCi/well of [3H]-Thymidine for 4 hours, and the level of DNA synthesis, as measured by
[3H]-Thymidine incorporation, was determined. For statistical analysis, the measurement of
[3H]-Thymidine incorporation as a function of NEUG concentration was modeled using a
four-parameter logistic model and EC50 values (pg/mL) were determined. Results for test
proteins are reported as a relative potency (RP%) value, generated by comparing EC50 value
of reference standard to the EC50 of the test sample analyzed within the same assay [RP% =
(EC50 reference/ EC50 sample)* 100].
[0156] The results (FIG. 12) demonstrate that activity of rHSA-G-CSF was better
maintained when incubated in low pH.
7. Effects of Salt concentration on aggregation of rHSA-G-CSF
[0157] The ionic strength of the rHA-G-CSF 60mg/ml solutions was controlled by altering
the amount of sodium chloride in the solution. The formulation buffer was PMTT10/7.2.
Samples were incubated for 1 day at 25°C/60% RH, at a sodium chloride concentration of
5mM, 10mM, 20mM, or 50mM, and subsequently tested by SE-HPLC.
[0158] The results show that increasing the salt concentration decreased rHSA-G-CSF
aggregation (FIG. 5).
8. Effects of phosphate concentration on aggregation of rHSA-G-CSF
[0159] The ionic strength of the rHSA-60mg/ml G-CSF solutions was controlled by
altering the amount of phosphate in the solution. The formulation buffer was PMTT pH 7.2.
Samples were incubated for 1 day at 25°C/60% RH, and at a phosphate concentration of
15mM, 20mM, 25mM, 30mM, 40mM, or 50mM and subsequently tested by SE-HPLC.
[0160] The results show that increasing the phosphate concentration decreased rHSA-G-
CSF aggregation (FIG. 6).
Example 5: Preparation of therapeutic NEUG-2

[0161] NEUG-2 was manufactured for clinical studies for use and administration at higher
concentrations. The formulation included NEUG at 50.0 mg/ml in PMMT20/6.0 (20 mM
sodium phosphate, 180 mM mannitol, 60 mM trehalose, 0.01% polysorbate-80).
[0162] Essentially, the same process of fermentation and purification was followed as
shown for NEUG-1 (see e.g., FIG 13).
[0163] NEUG-2 was prepared both as a liquid and in lyophilized form. The lyophilized
form was prepared to improve the robustness of the formulation for shipping and storage at
clinical sites, as well as to provide a stable product with an expected long shelf life. NEUG-2
was freeze-dried using the identical freeze-dry cycle as described for NEUG-1. The NEUG-2
formulation also produced well-formed, pharmaceutically elegant cakes.
Example 6: Comparison of NEUG-0, NEUG-1 and NEUG-2 Formulations
[0164] Table 3 shows the physiochemical comparability of NEUG-0, the standard lot of
NEUG-1 (reference standard 2378-R) and three different lots of NEUG-2. In the table, "CP"
means clear, pale yellow. "ND" means not determined.



[0165] Process related impurities (e.g., yeast host cell proteins) and product related variants
(SEC, RP, and IEC-HPLC) have been consistently reduced as the processes for
manufacturing NEUG-0 through NEUG-2 have been developed.
[0166] Tables 4 and 5 provide a summary of the formulation history and formulation
comparison of NEUG-0, NEUG-l and NEUG-2.

1. Effects of pH and phosphate concentration on monomer purity of rHSA-
G-CSF
[0167] The results displayed in FIG. 7, show monomer purity measured by SE-HPLC of
NEUG in PMTT10/7.2 and in PMTT20/6.0 (20mM Phosphate, 180 mM Mannitol, 60 mM
Trehalose Dihydrate, 0.01% (W/V) Polysorbate 80, pH 6.0) respectively. Samples contained
different protein concentrations at a range of 2.5 to 120 mg/ml, and were incubated at 25°C
for 24 hours prior to purity analysis.
[0168] Results show no significant reductions of purity were observed with rHA-G-CSF at
concentrations of up to 120 mg/ml in PMTT20/6.0 formulation buffer as measured by SE-
HPLC, whereas in PMTT 10/7.2 there was a 10% aggregation increase.
2. Effects of pH and phosphate concentration on aggregation of rHA-G-CSF
[0169] The results displayed in FIG. 8, show aggregation measured by SE-HPLC of 60
mg/ml rHA-G-CSF incubated at 4°C or 25°C /60% RH for a maximal period of 14 days in
PMTT20/6.0 or in PMTT10/7.2.
[0170] Results show no significant aggregation increase was observed after incubation at
25°C for 14 days for samples incubated in PMTT20/6.0 formulation buffer whereas samples
incubated under the same conditions in PMTT 10/7.2 formulation buffer showed a 6 %
increase in aggregation.
3. HPLC comparison of NEUG-1 versus NEUG-2
[0171] SEC-HPLC analysis and RP-HPLC analysis of NEUG-1 and NEUG-2 is shown in
FIG. 14A and 14B.
4. Charge comparability, peptide mapping and purity of NEUG-1 and
NEUG-2
[0172] The charge heterogeneity of NEUG-1 (referred to as "IP" in the figure) and NEUG-
2 (referred to as "2P" in the figure) were compared by IEC-HPLC. Results are shown in FIG
15A. The identity of the two peptides (NEUG-1, "IP" and NEUG-2, "2P") was also
compared by peptide mapping as shown in FIG. 15B.
[0173] Purity of NEUG-1 and NEUG-2 was also compared by SDS-PAGE and Coomassie
blue staining as shown in FIG. 16. The samples in each lane are shown in Table 6, below.

5. Freeze-Thaw
[0174] The effects of Freeze-Thaw were evaluated with NEUG-2 (in PMTT20/6.0) buffer
with concentration 15, 60,120 mg/ml. Samples were frozen and thawed from 0 to 10 times,
then analyzed via visual inspection and SEC, RP, IEC.
[0175] Results indicate that Neugranin does not appear to be sensitive to freeze-thaw
induced degradation for both old (PMTT10/7.2) and new (PMTT20/6.0) formulation buffers.
(Data not shown).
6. Short-term stability
[0176] Samples of NEUG (60 mg/ml) in old buffer (PMTT10/7.2) and new buffer
(PMTT20/6.0) were kept at 4 and 25°C /60% RH for one month, then tested by visual
inspection, SEC, RP, IEC and Bio-assay. Monomer purity was evaluated by SE-HPLC.
[0177] At 14 days, NEUG in the old buffer showed 6 % aggregates increased, while NEUG
in the new buffer showed no changes. There were no significant changes for one month by
visual, RP, IEC and Bio-assay.
7. Shaking
[0178] The effect of shaking-induced aggregation was evaluated with NEUG in old
formulation buffer (PMTT10/7.2) new formulation buffer (MPTT20/6.0) at three conditions

(15, 60,120 mg/ml), pH 6.0. Each sample was shaken horizontally at 120 rpm from 0 to 30
min at 25°C. Visual inspection and SE-HPLC were performed.
[0179] The Results of the SE-HPLC, RP-HPLC, IE-HPLC displayed no significant loss of
monomer. (Data not shown). Furthermore, the results from visual inspection indicate no
change in appearance as a result of shaking. This suggests that Neugranin may not be
sensitive to shaking-induced aggregation for both old and new formulation buffers.
8. Oxidation
[0180] Ion-exchange high performance liquid chromatography ("EEC") and reversed phase-
high performance liquid chromatography ("RPLC") can be used to monitor oxidation of
Neugranin. The experimental conditions for the hydrogen-peroxide study are as follows.
Neugranin at 8 mg/mL were exposed to 0.03% hydrogen peroxide and TBO. Samples were
analyzed by size exclusion chromatography ("SEC"), reverse phase ("RP"), and IEC after
incubation at 25°C for 0, 1, 3, 5, 8, 20, and 24 hours. For assessment of hydrogen peroxide
and TPB induced oxidation, RPLC, IEC were used. Chromatographs and data in tabular
form are shown in FIGS. 21 and 22.
[0181] HSA has been shown to oxidize in the presence of hydrogen peroxide. The sites of
oxidation identified include Cys-34, Met-123, Met-298, Met-446, and Met-548.
[0182] The chromatograms (FIG. 21) show the overlays for NEUG control (grey), after
exposure to hydrogen peroxide (light grey), and TPB (black) for 1 hour at RT. A significant
increase in a peak eluting 3 minutes after the main peak is shown in samples treated with
H2O2 by RP and EEC; samples treated with TPB show a peak eluting 3 minutes after the
main peak by RP and the peaks slightly increased and eluted 2, 3 minutes later than main
peak by IEC.
[0183] In summary, the results suggest that RP and EEC can detect different oxidized forms,
and both H2O2 and TBP could induce NEUG oxidations at which are methionine and cystein
residues. The oxidation of NEUG does occur under stressed conditions, but there is no
indication that it represents a major degradation pathway for NEUG upon storage and there
are no significant changes on their activities.
Example 8: Exemplary characterization of NEUG for pharmaceutical use

[0184] Three cGMP drug substance lots were produced using the NEUG-1 process and 5
lots were produced using the NEUG-2 process. In general, the NEUG-2 formulation yielded
protein of equal or better quality relative to NEUG-0 and NEUG-1. The test results for all
cGMP lots are provided in the tables in FIG. 17A-B. NEUG-1 lots are 060615001,
060628001, and 060707001. NEUG-2 lots are 071008005, 071025001, 071026004,
071106001, and 07116001. Testing of the lots was performed as follows.
1. Appearance by visual inspection
[0185] The appearance of Neugranin™ ("NEUG") was assessed by visual inspection using
forward illuminating fluorescent light against a black and white background.
2. pH
[0186] The pH meter was standardized using buffer solutions at pH 4.0, 7.0, and 10.0. After
standardization, the pH of a sample of NEUG was measured and recorded.
3. Osmolality by freezing point
[0187] The osmolality was determined by freezing point depression. Standardization was
performed using a 290 mOsm/kg calibration standard prior to measurement.
4. Concentration by measuring the absorbance at 280 nm
[0188] The concentration of NEUG was determined by measuring the absorbance at 280
nm. Protein concentration was calculated using the empirically determined extinction
coefficient of 0.54 mg-l*mL*AU.
5. Identity by ELISA
[0189] The identity assay used a standard sandwich ELISA format using methods well
known in the art. The assay was based on the specificity of antibodies for both the G-CSF
and albumin portions of the NEUG molecule. Together this antibody pair can distinguish
NEUG from G-CSF, HSA, or other albumin-linked proteins.
6. Purity by Coomassie stained SDS-PAGE
[0190] The purity of NEUG under denaturing conditions was evaluated using SDS-PAGE

with Coomassie stain. Samples were diluted in sample buffer with or without reducing agent
prior to analysis. Molecular weight markers and a NEUG reference standard were also
loaded onto each gel. After electrophoresis, the gel was stained with Coomassie and the
purity determined using densitometry. This method is used primarily to detect product-
related impurities such as dimers.
7. Purity by Silver Stained SDS-PAGE
[0191] The purity of NEUG under denaturing conditions was evaluated using overloaded
SDS-PAGE with silver staining. Samples were diluted in sample buffer with or without
reducing agent prior to analysis. Samples were loaded up to 10 µg/lane. Molecular weight
markers and a NEUG reference standard were also loaded onto each gel. After
electrophoresis, the gel was stained with silver. Comparisons between migration distances
and band patterns are used to determine comparability between NEUG samples and reference
standard. This method was used to detect low levels of product and process-related
impurities.
8. Purity by Size Exclusion HPLC
[0192] The purity of NEUG under native conditions was evaluated using size exclusion
HPLC. Samples were analyzed using a TosoHaas G3000SWXL column (7.8 mm x 30 cm)
with an isocratic mobile phase consisting of phosphate and sulfate. Purity of NEUG was
determined by calculating the ratio of the area under the main peak to the total peak area at
280 nm. This assay detects product-related sizing impurities, including dimers and
aggregates.
9. Purity by Reversed Phase HPLC
[0193] Reversed phase HPLC was used to measure the purity of NEUG. Samples were
analyzed using an Agilent Zorbax reversed phase column (StableBond C8, 300Å, 5µm, 250 x
4.6 mm) with an elution gradient of increasing acetonitrile concentration. Purity was
determined by calculating the ratio of the main peak area to total relevant peak area at a
detection wavelength of 215 nm.
[0194] The peak preceeding the main peak in the RP-HPLC assay has a mass consistent
with a Neugranin variant containing a di-hexose modification at Thr-133 of the G-CSF

moiety. This modification does not affect potency and was present at consistent levels in all
development, toxicology, and cGMP batches manufactured to-date. Thr-133 is a known site
for modification of G-CSF.
10. Potency by bioassay (NFS-60 cell proliferation)
[0195] The NEUG potency assay is a cell based proliferation assay, based on the induction
of NSF-60 cell proliferation. NFS-60 cells proliferate in response to both G-CSF and NEUG.
In this assay, 10,000 cell/well were plated in a 96-well microplate and were treated with
NEUG proteins for 20 hours at 37°C. After a 20-hour incubation the NFS-60 cells were
pulsed with 0.5 uCi/well of [3H]-Thymidine for 4 hours, and the level of DNA synthesis, as
measured by [3H]-Thymidine incorporation, was determined. For statistical analysis, the
measurement of [3H]-Thymidine incorporation as a function of NEUG concentration was
modeled using a four-parameter logistic model and EC50 values (pg/mL) are determined.
Results for test proteins were reported as a relative potency (RP%) value, generated by
comparing EC50 value of reference standard to the EC50 of the test sample analyzed within
the same assay [RP% = (EC50 reference/ EC50 sample)* 100].
11. Residual DNA by Threshold Method
[0196] The method for measuring residual DNA in samples is based on a commercially
available kit (Molecular Devices Total DNA Threshold DNA quantification kit). The sample
was initially diluted, protein digested, and the DNA extracted. The concentration of DNA
was then quantified using the Threshold kit. This kit immuno-labels, immobilizes, and
electrochemicaUy measures the concentration of DNA relative to a calf thymus DNA
standard curve. The results were internally qualified by spike recovery of DNA standard
within acceptable limits.
12. Endotoxin by Kinetic Turbidimetric Method
[0197] Endotoxin concentrations in NEUG solutions were determined using a standard
automated kinetic turbidimetric endotoxin analysis system. The kinetic turbidimetric system
is a refinement of the gel-clot method, and measures turbidity increases that precede gel-clot
formation. Limulus amoebocyte lysate (LAL) was added to the standards and test articles and
incubated at 37°C. During incubation, the turbidity of the reaction mixture was monitored

spectrophotometrieally. Turbidity was proportional to the endotoxin concentration. The rate
of turbidity formation in the test article is compared with the turbidity formation in the
standard curve of known standard concentrations.
13. Biobmrden by membrane filtration
[0198] The membrane filtration method is similar to the United States Pharmacopoeia (USP
<61>) Microbial Limits Test that estimates the number of viable organisms present in a
sample. In the membrane filtration method, the organisms are captured out of the test solution
on a membrane filter, which is then plated on a suitable growth medium. A sample volume
of 10 mL of test article is diluted in 90 mL of sterile phosphate buffered saline (PBS) prior to
testing. The entire 100 mL of diluted test article is filtered. After the filtration step, the filter
membrane is transferred to Heterotrophic Plate Count (HPC) agar and incubated at 30 to
35°C for 2 to 3 days. The HPC agar differs from the Soybean Casein Digest Agar (SCD,
which is also called TSA) cited in USP <61>. HPC agar is designed to recover a broad range
of aerobic, heterotrophic bacteria, as is TSA, However, HPC agar is also designed to
maximize recovery of stressed organisms.
14. Tungsten Study
{0199] It is known that certain syringe brands contain tungsten as an excipient. Liquid
NEUG samples were tested in the presence of various forms of tungsten (sodium tungstate
dehydrate, sodium polytungstate, ammonium tungstate, and tungsten oxides WO3 and WO2)
at various concentrations (500 ppb, 1000 ppb and 2500 ppb) at 2-8°C and 25 °C for up to
three months. Samples were analyzed by SE-HPLC, RP-HPLC and bioassay. There were no
significant effects of tungsten on NEUG samples after three month stability studies.
15. Characterization Assays
[0200] The following analytical were also used for characterization information.
N-terminal sequence
[0201] The amino acid sequence at the N-teiminas of Neugranin was identified using a PE
Applied Biosystems Procise 494 cLC protein sequencer. The method incorporates Edmam
degradation chemistry and utilizes gas-phase blot cycles, an on-line PTH-amino acid HPLC
analyzer, and SequencePro data analysis software.

Charge Heterogeneity by IE-HPLC
[0202] IEG-HPLC was used to measure the relative abundance of charge heterogeneity
present in NEUG, Samples were analyzed using a Dionex Pro-Pac Wax 10-ion exchange
column (250 x 4.6 mm) with mobile phases consisting of Bis-Tris and NaCl. This method has
demonstrated sample-independent variability and at this point in development is used as a
qualitative rather than quantitative assay.
Peptide mapping
[0203] Peptide mapping is used to determine the structure integrity of NEUG, Peptide
mapping was performed using Lys-C for digestion and reversed-phase HPLC with gradient
elation for peptide separation. The protein sample was first reduced and denatured and then
alkylated, A buffer exchange was carried out, followed by a 1 hour, then overnight, Lys-C
digestion. The Lys-C peptide mixture resulting from the digestion was separated by reversed-
phase HPLC to give a peptide profile using an YMC Pack ODS-A C18 column (4.6 mm x
250 mm) with an Agilent 1100 HPLC system. The HPLC elution was monitored using a
diode array UV detector and Finigan LCQ DUO- Ion Trap mass spectrometer with
electrospray ionization. Mass spectrometry data were processed using Xcalibur V2.3
software. The mass accuracy of this method was less than 20 ppm. The identities of HPLC
peaks were established by LC-MS.
Electrospray Ionization Mass Speetrometry
[0204] Mass analyses were performed using an Applied Biosystems QStar ESI-Quadrapole
Time of Flight (QTOF) Pulsar-i mass spectrometer equipped with a Pico View nano-
electrospray source. Samples were introduced into QStar by Hamilton syringe and Harvard
syringe pump. The electrospray ion source was operated at 2500V with a curtain gas value of
25. The instrument was calibrated with myoglobin. The mass to charge ratios were recorded
by scanning the full mass range (500-3000 m/z) within the mass analyzer every second
throughout the allotted time of analysis. The resulting total ion current was analyzed using
Analyst v1.4 software. The mass accuracy of the instrument is less than 10 ppm.
Residual S. cerevisiae (veast) Host Cell Protein
[0205] The amount of yeast host cell protein (yHCP) present in the bulk was determined

using an ELISA based on custom antibodies raised by immunization with host cell proteins
derived from a null strain of the same yeast host used to express NEUG.
Free thiol by Ellman's Assay
[0206] Ellman's reagent, 5,5'-Dithio-bis-(2-nitrobenzoic acid) (DTNB) was used to
determine the free thiol content in NEUG samples. DTNB reacts with free sulfhdryl groups
to yield a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB). TNB absorbs strongly at
412nm allowing the reaction to be easily quantitated by comparison to a N-acetyl Cysteine
(NAC) standard curve. To increase the accuracy of the method, the reaction was carried out
under partially denaturing conditions. This allows the DTNB access to free sulfhdryl groups
that may not be folly exposed under native conditions.
16. Stability
[0207J Bulk drug substance ("BDS") is stored and shipped at -80°C (nominal value,
acceptable temperature is < -65°C). BDS stability was studied by storing the product at the -
SOT and accelerated storage conditions. The reference standard (NEUG-1, Lot 2378-R) was
placed on stability first to provide leading stability for GMP BDS and an understanding of the
stability of the reference standard itself. Typically for each process (NEUG-1, NEUG-2) at
least one development and one cGMP lot was placed on stability, with each study detailed in
a study-specific protocol that follows ICH guidelines. Standard accelerated conditions were
used to define assay suitability and likely degradation pathways, as well as help predict
expiration. The stability was monitored by appearance, pH, protein concentration,
osmolality, SEC-HPLC, RP-HPLC, SDS-PAGE, and bioassay.
[0208] A summary of the representative stability results for NEUG 1 reference standard lot
2378 is summarized below in Tables 7-9 below.

[0209] No significant changes in any parameter were seen out to 36 months at the
recommended storage condition, out to 18 months at -20°C or out to 12 months at 2-8°C.
[0210] 1 month data is shown for the NEUG-2. No significant changes under the
recommended storage conditions, at -20°C or at 2-8°C are noted. Table 10.



[0211] Stability data for two lots of NEUG-2 in liquid form at 2-8°C are shown below in



[0212] As described above, changes to the formulation were made from the NEUG-1
process to the NEUG-2 process. These changes were made to ensure product quality at a
higher NEUG concentration and did not include any new excipients. Primary changes were
an increase in the ionic strength (from 10 to 20 mM) and a reduction in the pH (from 7.2 to
6.0). The BDS formulation has been improved from the NEUG-1 to NEUG-2 process to
reduce concentration dependent aggregation. It is thus expected that the NEUG-2
formulation will demonstrate improved stability relative to the already robust NEUG-1.
[0213] In addition, there were no significant effects of tungsten on NEUG-2 after three
month stability studies either in lyophilized for or in liquid form at recommended storage
conditionas at 2-8°C (analysis via SE-HPLC, RP-HPLC and potency, data not shown).
Example 9: Description and Composition of Exemplary Drug Product: NEUG-2
1. Description of dosage form
[0214] In one embodiment, Neugranm™ ("NEUG") is supplied as a sterile, lyophilized
formulation in single-use Type 1 glass vials, which are sealed with a coated rubber stopper
and a flip-off seal. NEUG can be stored at 2-8°C. Upon reconstitution with 1 .0mL of Sterile
Water for Injection (WFI), each vial contains 50 mg/mL (50mg/vial deliverable) NEUG in
20mM sodium phosphate, 180 mM manttitol, 60 mM trehalose dihydrate, 0.01% (w/v)
polysorbate 80, pH 6.0. After reconstitution the vial can be held at room temperature and
should be used within 8 hours.
2. Quantitative composition

[0215] The quantitative composition of one exemplary NEUG drug product (NEUG-2) is
provided in Table 11, below. Note that the liquid form of NEUG-2 has the same quantitative
composition as the lyophilized form. The liquid form can be provided in a prefilled syringe
(see section 9, below).

3. Reconstitution buffer
[0216J Sterile Water For Injection (WFI) can be used for reconstitution of NEUG in
lyophilized form.
4. Excipients
[0217] The composition of the exemplary NEUG formulation is 2.77 mg/mL sodium
phosphate, 33.79 mg/mL mannitol, 22.7 mg/mL trehalose dihydrate, and 0.1 mg/mL
polysorbate 80, pH 6.0 (see Table 5 above). The sodium phosphate is a buffering agent,
mannitol is used as a bulking agent to provide good cake structure and to adjust tonicity,

trehalose is used as a cryoprotectant, and poiysorbate 80 is used to reduce the potential for
aggregation and adsorption, At the concentration of active pharmaceutical ingredient ("API")
in NEUG-2 of 50 mg/mL, the protein itself acts as a robust buffer. Compendial excipients
used for the formulation comply with multiple-compendia (MC). The exception is trehalose
dihydrate for which no compendia is currently available; however, it complies with a rigorous
non-compendial set of specifications. Trehalose is GRAS listed at the US FDA and is used in
the formulation of numerous commercial parenteral pharmaceuticals (for example,
bevacizumab (Avastin) and trastuzumab (Herceptm)),
5. Formulation development
[0218] The initial formulation used for toxicology studies was designed to support storage
as a frozen liquid at -20°C (NEUG-0). To improve me robustness of the formulation for
shipping and storage at clinical sites, as well as to provide a stable product with an expected
long shelf life, a lyophilized form has been used for all clinical material to date. The initial
clinical material (NEUG-1) has proven quite stable, with a current shelf-life of 2 years, which
will likely be further extended. Subsequent formulation development demonstrated that
higher ionic strength and lower pH further stabilized the API at higher concentration (> 25
mg/mL). To this end, one formulation (NEUG-2) has a lower pH (6.0 vs 7.2) and higher
phosphate concentration (20 vs. 10 mM). Forced degradation studies demonstrate that this
formulation protects the drug substance in the liquid state from vigorous shaking, repeated
freeze-thawing, and concentration induced aggregation. The identical freeze dry cycle as
described for NEUG-1 was used for the NEUG-2 formulation; both produced well-formed
cakes.
6. Manufacturing process development
[0219] In some embodiments, NEUG final drug product is generated by diluting bulk drug
substance with formulation buffer to the desired concentration (50 mg/mL), filling, and then
freeze drying the product,
{0220] To support comparability between the toxicology and clinical material, a lot of final
drug product ("FDP") from both processes (NEUG-1 and NEUG-2) was characterized
physicochemically.

[0221] The results of the physicochemicai comparability study are included in Table 12
below, and demonstrate that the quality of the NEUG-2 product is at least as good, if not
better, than the NEUG-1 product. The freeze-drying process does not have any adverse
product impact.

[0222] The NEUG used for initial clinical studies (NEUG-1) is physicochemically
comparable to the currently formulated NEUG (NEUG-2). The changes made to the
manufacturing process and dosage form did not have an impact on clinical efficacy or safety
(data not shown).

7, Batch analysis
[0223] FIG, 20 summarizes the results of analysis performed on a representative,
development lot of NEUG-2 final drug product. In the table, "ND" means the test was "not
done" for development lots.
8, Stability of final drug product
[0224] A stability study for a lot of the final drug product of NEUG-2 was initiated, At the
one month time point, no significant changes were observed for any samples. (Data not
shown). Stability conditions were as follows; NEUG-2 development lot at 50.0 mg/ml
NEUG in PMTT10/6.0; 1.2 mls liquid in a 3 ml glass vial; storage at 2-8°C, 25°C/60% RH, or
40°C/75% RH.
9, Studies of NEUG-2 liquid in prefilled syringes
[0225] To assess the feasibility of providing NEUG-2 (PMTT, pH 6.0) as a liquid in a
prefilled syringe for commercial products, the following studies were performed.
[0226] It is known that the some syringes (e.g., from Becton Dickinson) contain two major
excipients, tungsten and silicone, which might interact with the product and/or the buffer. To
evaluate the interactions between the syringe excipients and the product, several studies were
carried out including a silicone oil migration study, silicone-spiked-in and tungsten-spiked-in
studies, as well as the long-term stability studies of the prefilled syringes. The methods used
to assess the stability of CO 10639 (NEUG-2) in a liquid state included visual inspection, SE-
HPLC, RP-HPLC, IE-HPLC, Bioassay, etc.
[0227] To summarize the results, no significant changes were found in the samples spiked
with five forms of tungsten (sodium tungsten dehydrate, sodium polytungstate, ammonium
tungstate, W03 and W02) at three different doses (500,1000 and 2500 ppb) and kept at
either 2-8°C or 25°C over three months. Samples were analyzed via SE-HPLC, RP-HPLC,
and bioassay (data not shown),
[0228] In the silicone compatibility study, there were no effects of silicone on the product
quality observed when samples were in syringes coated with two different doses of silicone
(0.4 mg or 0.8 mg) and kept at 2-8°C for 4 weeks, or samples were spiked in with two
different doses of silicone and kept at 2-8 °C for three months (data not shown). The silicone
oil distribution in both formulation buffer and drug product solution were compatible and

remained essentially even (data not shown). There were no gliding force changes on prefilled
syringes after two months' storage (data not shown). Moreover, CG10639 appeared to be
insensitive to agitation and needle-shear (data not shown).
[0229] The long-term stability studies for at least up to nine months demonstrate that liquid
NEUG-2 is stable in the prefilled syringe at 2-8 °C, is stable for at least up to 18 months at
4°C, and is compatible with the materials of product contact. Data for the long term stability
of liquid NEUG-2 in prefilled syringes is shown in Table 13, below.

[0230] Based on these studies, there are no significant interactions between tungsten or
silicone and the NEUG product with PMTT formulation (20 mM Phosphate, 180 mM
Mannitol, 60 mM Trehalose Dihydrate, 0.01% (W/V) Polysorbate 80, pH 6.0).
Clinical evaluation of NEUG in human patients
[0231] The following examples are provided in two main sections entitled "Phase I" and
"Phase II" Each phase includes two parts, Part A and Part B. The Phase I and Phase II
examples are summarized in Table 14 below. Note that NEUG-1 was tested in Phase I, and
NEUG-2 was tested in Phase II.



[0232] Each Phase is divided into five sections: 1) objectives, 2) patient characteristics, 3)
study agent, 4) study characteristics, and 5) results of Parts A and B.
Example 10: PHASE I
1. Objective
[0233] The Phase IA/B, IIA/B study was performed to evaluate the safety, tolerability,
immunogenicity, pharmacokinetics and pharmacodynamics of subcutaneously administered
Neugranin™ ("NEUG") (recombinant human aibumin-human granulocyte colony stimulating
factor) in subjects receiving myelosuppressive chemotherapy (doxorubicin/docetaxel).
[0234] For Phase I, the primary study objectives were to assess the safety profile of NEUG
given subcutaneously over a range of potential therapeutic doses compared to pegfilgrastim
by measuring the frequency, severity, and duration of treatment-emergent adverse events and
correlating them with the time and dose of NEUG administration.
[0235] Secondary study objectives were to assess the pharmacokinetics and
immunogenicity of NEUG, and to compare the effect of NEUG administration on the
incidence, severity and duration of neutropenia to pegfilgrastim in patients receiving
doxorubicin/docetaxel.
[0236] Phase I was performed as two parts, Part A and Part B as noted in Table 2 above.
2. Patient characteristics

[0237] For Phase I, patients were screened based on the following characteristics or
parameters:
[0238] Inclusion:
1. Patients with histologically-confirmed breast cancer scheduled to receive
doxorubicin and docetaxel.
2. 18 years of age or older.
3. Adequate hematologic function.
4. ANC > 1500/mm3
5. Platelets > 100,000/mm3
6. Adequate hepatic and renal function:
7. Serum creatinine < 2.0 x upper limit normal
8. Total bilirubin within normal limits (WNL) for local laboratory
9. Serum transaminases (SGOT/SGPT) < 1.5 x upper limit normal
10. Alkaline phosphatase < 2.5 x upper limit normal
11. ECOG performance status 0 or 1.
12. Eligible to receive doxorubicin based on a left ventricular ejection fraction
(LVEF) within normal limits.
13. Have the ability to understand the requirements of the study, provide written
informed consent (including consent for use and disclosure of research-related
health information) and comply with the study protocol procedures.
[0239] Exclusion:
1. More than 1 prior chemotherapy regimen (including adjuvant therapy if given
within the last 12 months); any chemotherapy/immunotherapy within 4 weeks
prior to study entry; cumulative anthracycline dose that would preclude 2 full-
dose cycles of doxorubicin in this study.
2. Prior use of any nitrosoureas (BCNU, CCNU) or mitomycin-C within 6 weeks
of study chemotherapy.
3. Cardiac history, signs or symptoms that, in the Investigator's opinion,
preclude the use of an anthracycline-based chemotherapy regimen.
4. Prior surgery or radiation therapy within 2 weeks of study chemotherapy.

5. Prior wide field irradiation to the pelvis or to greater than 20% of the marrow-
bearing areas, or bone marrow involvement.
6. Prior high-dose chemotherapy with hematopoietic stem cell transplant.
7. Prior use of myeloid (G-CSF or GM-CSF) growth factors within 4 weeks of
study chemotherapy.
8. Prior use of erythropoietin within 4 weeks of study chemotherapy.
9. History of myeloid malignancy or myelodysplasia.
10. Known brain metastases unless adequately treated (surgery or radiotherapy),
no evidence of progression with a minimum of 3 weeks observation and
neurologically stable off anticonvulsants and steroids.
11. Known sickle cell disease.
12. Diagnosis of adult respiratory distress syndrome (ARDS).
13. Current infection requiring intravenous or oral antibiotics.
14. Known history of allergies to yeast-derived products.
15. Known hypersensitivity to E coli-derived proteins, pegfilgrastim, filgrastim,
or any other component of pegfilgrastim (phase 2 only).
16. Pregnant female or nursing mother (over the course of the study, all females
must practice a method of contraception with greater than 90% reliability, or
be sterile or postmenopausal).
17. Known HIV positive or active hepatitis (patients with unknown status will not
be tested).
18. Males who do not agree to use effective contraception throughout the study
and for a period of 30 days after the last dose of study agent.
[0240] Subjects were removed from further treatment for the following reasons:
1. Disease progression
2. Unacceptable toxicities despite optimal treatment
3. Intercurrent illness at the investigator's discretion
4. Doxorubicin regimen - Maximum lifetime permissible cumulative dose
reached (see eligibility criteria)
5. Withdrawal of consent
6. Non-compliance/Loss to follow-up

7. Pregnancy
[0241] If treatment with NEUG was stopped, subjects remained on study and were
followed at least 30 days following the final dose of any study drug for scheduled safety and
PK assessments.
3. Study agent
[02421 NEUG (recombinant human albumin-human granulocyte colony stimulating factor,
rHSA-G-CSF), is a fusion protein with a molecular mass of approximately 85kDa connected
in a single chain comprising residues 1-585 corresponding to the mature form of HSA and
residues 586-759 corresponding to the mature form of human G-CSF. The therapeutic
moiety of NEUG is recombinant human DNA-derived G-CSF.
[0243] NEUG was supplied as a sterile, lyophilized formulation in single-use Type 1 glass
vials and stored at 2-8°C. Upon reconstitution with 1.1 ml of sterile water for injection, each
vial contained 15mg/ml (15mg/vial deliverable) NEUG in 10 mM sodium phosphate, 200mM
mannitol, 60mM trehalose dehydrate, 0.01% (w/v) polysorbate 80, pH 7.2.
[0244] The composition of the NEUG drug product used in Phase I is presented in Table 1.
[0245] Commercially available Neulasta® (pegfilgrastim) was supplied in 0.6 ml prefilled
syringes for subcutaneous injection. Each syringe contains 6 mg pegfilgrastim (based on
protein weight), in a sterile, clear, colorless, preservative-free solution (pH 4.0) containing
acetate (0.35 mg), sorbitol (30.0 mg), polysorbate 20 (0.02 mg), sodium (0.102 mg) in water
for injection. USP.
[0246] NEUG (50,150,300 or 450 µg) or Neulasta® (pegfilgrastim) (6 mg) was
administered by subcutaneous administration.
4, Study characteristics
a. Study schedule and duration
[0247] This study was a first-in-man, multi-center, open-label non-controlled sequential
dose escalation of a followed by a controlled, randomized trial conducted in 62 subjects with
breast cancer scheduled to receive doxorubicin/docetaxel. The study consisted of 2 parts.
Part A was a sequential dose escalation in 13 subjects, 4 dose cohorts (50,150, 300, or 450
µg/kg) with 3 subjects in each of the 50,150 and 450 µg/kg cohorts and 4 subjects in the 300
jµg/kg cohort, to evaluate safety prior to the randomized, Part B of the trial.

[0248] In Part A, subjects received the first dose of NEUG at least 2 weeks prior to the start
of chemotherapy (cycle 0) for an initial assessment of safety and effects on absolute
neutrophil count ("ANC") in the absence of cytotoxic chemotherapy. After a minimum of 2
weeks follow-up, subjects received NEUG at the same dose following chemotherapy in
cycles 1 and 2 if there were no dose-limiting adverse events considered related to NEUG in
cycle 0 and the subject continued to meet all eligibility criteria.
[0249] In Part A, dose limiting toxicity (DLT) was defined as grade 2 or greater clinically
significant adverse event(s) considered possibly, probably or definitely related to the study
agent with the exception of grade 2 bone pain. Within each Part A cohort, the initial study
drug administration to each subject entering the trial was separated by a minimum of 24
hours to monitor for acute adverse events.
[0250] The decision to escalate to the next dose level was based upon the review of the
safety data for at least 7 days after the first dose administration of NEUG for all subjects in a
given cohort. If none of the 3 subjects experienced a DLT, dose escalation continued with
the enrollment of 3 subjects at the next dose level. If 1 of 3 subjects in a given cohort
exhibited evidence of a DLT, another 3 subjects were recruited at that dose level for a total of
6 subjects per cohort. Dose escalation continued to occur if only 1 of 6 subjects experienced
a DLT. If 2 of 6 subjects develop a DLT, dose escalation stopped and no further NEUG
treatments were administered.
[0251] The remaining subjects completed their scheduled safety, pharmacokinetic and
pharmacodynamic evaluations.
[0252] Following demonstration of safety in the initial Part A cohorts, Part B was
performed. In Part B, subjects were randomized in a parallel fashion to 1 of 3 treatment
groups: NEUG 300 µg/kg (n = 20), NEUG 450 µg/kg (n = 21), or pegfilgrastim (n = 10) at
the approved dose of 6 mg administered approximately 24 hours after study chemotherapy.
[0253] Tables 15 and 16 below summarize the disposition of the subjects in Phase I, Parts
A and B. FIG. 26 shows the chemotherapy cycles for Phase I study, Parts A and B.



b. Concomitant therapy during Phase I, Parts A and B
Chemotherapy
[0254] The chemotherapy regimen for this trial consisted of doxorubicin 50 mg/m2 and
docetaxel 75 mg/m administered sequentially by intravenous infusion on day 1 of treatment
for up to two 21-day cycles.
[0255] Prior to receiving each cycle of therapy, subjects had to have an absolute neutrophil
count (ANC) > 1.5 x 109/L and platelets > 100 x 109/L. Treatment could be delayed up to
two weeks for hematologic recovery.
[0256] The combination of doxorubicin and docetaxel has been reported to have significant
clinical activity in patients with breast cancer. However, the combination is highly
myelosuppressive with higher rates of grade 3 or 4 neutropenia than other standard regimens.
[0257] Even with the addition of CSFs, the combination of doxorubicin and docetaxel has
induced grade 4 neutropenia in 79% of patients and febrile neutropenia rates of 9-18%. This
doxorubicin/docetaxel regimen has been used in studies of new agents to prevent neutropenia

and its complications. Therefore, the combination of doxorubicin and docetaxel is an
appropriate chemotherapy regimen to study the potential of a new agent like NEUG.
Doxorubicin
Pharmacologic Data
[0258] Doxorubicin hydrochloride is an anthracycline antibiotic obtained from
streptomyces peucetius var caesius which inhibits DNA and DNA-dependent RNA synthesis,
as well as protein synthesis. Doxorubicin is active in all phases of the cell cycle but
maximally cytotoxic in S phase. Excretion of the drug is predominately by the liver; renal
clearance is minor.
Pharmaceutical Data
[0259] The drug is marketed commercially in 10, 20 50,100 or 200 mg vials. Lyophilized
preparations may be reconstituted with sterile water for injection, dextrose 5% solution, or
0.9% saline for injection.
Side Effects and Toxicity
[0260] Myelosuppression, primarily leukopenia, with a nadir of approximately 10-14 days,
and cardiotoxicity, including a rare, acute pericarditis- myocarditis syndrome and a delayed,
cumulative dose related cardiomyopathy are the dose-limiting toxicities of doxorubicin.
Marked alopecia and moderate nausea/vomiting are expected toxicities. Extravasation
reactions producing local skin and tissue damage at the site of inadvertent extravasation,
stomatitis, hyperpigmentation of the skin (particularly the nailbeds), and a "recall"
phenomenon at sites of previous irradiation have been reported.
Docetaxel
Pharmacologic Data
[0261] Docetaxel is a semisynthetic taxoid that binds to free tubulin and promotes assembly
of stable microtubules, interfering with mitosis and cell replication (cell cycle specific for M
phase). Docetaxel is extensively protein-bound, extensively metabolized in the liver, with
fecal excretion of approximately 75% of the dose within 7 days.
Pharmaceutical Data

[0262] Docetaxel (Taxotere™, Sanofi Aventis) is provided in 80mg/2 mL or 20 mg/0.5 ml
single-dose vials with an accompanying diluent (13% ethanol in Water for Injection) vial.
Each ml of Taxotere contains 40 mg of docetaxel (anhydrous) and 1080 mg polysorbate 80.
Side Effects and Toxicity
[0263] Docetaxel should not be given to patients who have a history of severe
hypersensitivity reactions to docetaxel or other drugs formulated with polysorbate 80 such as
etoposide and vitamin E.
[0264] Patients who experience severe hypersensitivity reactions should not be
rechallenged. Patients receiving docetaxel should be premedicated with corticosteroids as
outlined below.
[0265] Mild to moderate liver impairment results in delayed metabolism by 27% and a 38%
increase in systemic exposure (AUC). Docetaxel should not be given to patients with SGOT
and/or SGPT > 1.5 times normal limits and alkaline phosphatase > 2.5 times normal limits.
Fluid retention occurred in 17% (moderate) and 6% (severe retention) of patients in Phase in
studies despite corticosteroid premedication. Severe neurosensory symptoms (paresthesia,
dyesthesia, pain) have been observed.
[0266] Expected side effects include myelosuppression, primarily leukopenia, with a nadir
of approximately 9 days with recovery by day 15-21. Alopecia, nail and cutaneous changes,
stomatitis, myalgia/arthralgia, nausea/vomiting, and hypotension have been reported.
Chemotherapy Dosage, Administration and Dose Modifications
[0267] On day 1 of each treatment cycle, chemotherapy (doxorubicin followed by
docetaxel) was administered.
[0268] Doxorubicin was administered at a dose of 50 mg/m2 by IV bolus through the side
arm of an infusing intravenous line or central venous catheter to avoid extravasation injury.
[0269] Docetaxel 75 mg/m2 was diluted in 250 mL 0.9% saline or 5% dextrose solution and
administered intravenously over approximately 1 hour via a polyethylene-lined infusion set.
Vital signs were obtained immediately prior to and after the end of the docetaxel infusion.
[0270] Prior to receiving each cycle of therapy, subjects had to have an absolute neutrophil
count (ANC) > 1500/mm3 and platelets > 100,000/mm3. Treatment could be delayed up to
two weeks for hematologic recovery. A 25% dose reduction of chemotherapy doses was

allowed for grade 3-4 non-hematologic toxicities, two grade 3-4 infectious episodes, or grade
4 thrombocytopenia.
[0271] Subjects experiencing severe hypersensitivity reactions or non-hematologic
toxicities that preclude further cycles of chemotherapy were removed from study treatment
but completed follow-up.
Chemotherapy Pre-medication
[0272] Oral (IV as needed) corticosteroids (such as dexamethasone 8mg BID) were
administered for three days starting 1 day prior to docetaxel administration in order to reduce
the incidence and severity of fluid retention and hypersensitivity reactions.
[0273] The use and selection of anti-emetic agents or other pre-medications (e.g. H2
antagonists) was left to the discretion of the treating physician.
Prohibited Medications
[0274] Subjects should not have received any of the following medications and or
procedures during this study and for the additional times specified below:
1. Other investigational agents within 30 days of initiating study agent and for
the duration of the trial.
2. Subsequent cycles of chemotherapy should not be initiated until 14 days
following dosing with NEUG.
3. Cytokines, other hematopoietic growth factors and prophylactic antibiotics for
the duration of the trial unless prolonged or febrile neutropenia occurs. If the
subject was treated with G-CSF at any time between the screening period and
Day 0 they were not eligible to receive NEUG and were discontinued from the
study.
Allowed Medications
[0275] Subjects were allowed to continue their baseline medications(s). The daily dose of
each medication was maintained throughout the study if possible. If for any reason deemed
necessary by the investigator, a subject required additional medication(s) or change of dose,
the medication(s), route of administration, and the indication for which it was given was
recorded on the appropriate pages of the CRF.
Antibiotics

[0276] All subjects received prophylactic oral antibiotics (e.g. ciprofloxacin) following
each course of chemotherapy to reduce the likelihood of infection. If febrile neutropenia or
persistent severe neutropenia (ANC < 0.5 x 109/L for > 5 days) occurred, the subject was
considered a treatment failure, removed from the study, completed study follow-up and
received all standard supportive care, including growth factor support at the Investigator's
discretion.
[0277] Subjects who experienced severe hypersensitivity reactions or non-hematologic
toxicities that precluded further cycles of chemotherapy were also removed from study
treatment and completed follow-up.
c. Safety assessments
[0278] The safety of NEUG was assessed by evaluation of the type, frequency, and severity
of adverse events ("AEs"), changes in clinical laboratory tests (hematology and clinical
chemistry), immunogenicity, physical examinations, and the monitoring of vital signs over
time. All AEs and laboratory toxicities were graded based on the National Cancer Institute
Common Terminology Criteria for Adverse Events (NCI-CTCAE Version 3.0,12 December
2003). Adverse events (to include serious adverse events, "SAEs") were captured from the
start of study drug administration through 30 days following the final dose of any study drug.
Laboratory assessments were obtained as outlined in the Schedule of Assessments. In the
event of any Grade 4 neutropenia toxicity, labs were obtained every day until ANC > 500. If
the subject's next cycle of therapy was delayed (and after the last cycle of treatment),
complete blood count (CBC) with differential was obtained at least twice weekly until ANC
> 1500.
5. Results of Phase I. Parts A and B
a. General
Statistical Methods:
[0279] The data related to safety, pharmacokinetics (PK), pharmacodynamics (PD) and
immunogenicity parameters were analyzed using descriptive statistical methods.
[0280] For frequency and severity of adverse events, and for laboratory toxicity grading,
counts and rates are presented.

[0281] Efficacy analyses included the incidence and duration of grade 4 and grade 3-4
neutropenia, nadir ANC, time to nadir ANC, time to recovery (to ANC > 0.5 x 109/L and
ANC > 1.0 x 109/L), and the incidence of febrile neutropenia.
[0282] No strict statistical power requirement was used to select the sample size for this
study. A study with a power of 80% to demonstrate non-inferiority of NEUG to
pegfilgrastim at a significance level of 5% was calculated to require approximately 37
subjects per treatment arm. As this was a phase l/2a study conducted primarily for safety, it
was determined that the required sample size to be powered for effect was larger than
appropriate. As such, efficacy trends were evaluated.
Disposition/Demographics:
[0283] A total of 13 subjects were enrolled in the Part A, sequential dose escalation portion
of the trial. A total of 51 subjects were enrolled in the Part B portion, and randomized to
NEUG 300 µg/kg (n=20), NEUG 450 µg/kg (N=21), or pegfilgrastim 6 mg(n=10).
b. Study results
[0284] In initial dose-finding, in the absence of chemotherapy, NEUG was well tolerated
and resulted in the expected rise in ANC, which peaked between days 2 and 4 and returned to
normal by day 14 (FIG. 23).
[0285] In Part A, all three subjects in the 50 µg/kg NEUG dose group and 1 subject in the
450 µg/kg Neugranin dose group experienced febrile neutropenia or severe neutropenia
lasting greater than 5 days. In Part B, one subject in the 300 µg/kg NEUG dose group and 2
subject in the 450 µg/kg NEUG dose group experienced febrile neutropenia or severe
neutropenia lasting greater than 5 days. One subject in the pegfilgrastim group experienced
febrile neutropenia or severe neutropenia lasting greater than 5 days.
c. Immunogenicity
[0286] Serum samples for antibodies to NEUG were obtained prior to dosing on Day 1 of
every NEUG cycle and at the end of treatment visit (at least 15 days after the last dose) in
subjects receiving NEUG. If at any time during the study a subject developed a positive anti-
NEUG antibody response, a repeat sample was obtained approximately 6 months after the
final NEUG dose.

[0287] Testing was completed on all subjects through the end of treatment for both Part A
and B. All samples were negative for antibodies to NEUG.
d. Adverse events
[0288] During Part A, dose-limiting toxicity (DLT) was defined as grade 2 or greater
clinically significant adverse event(s), considered possibly, probably or definitely related to
the study agent with the exception of grade 2 medullary bone pain.
[0289] No DLT was encountered in cycle 0 in any of the Part A cohorts. Only 2 adverse
event were reported as related to NEUG administration: bone pain and exacerbation of pre-
existing hypertension, the latter occurring 7 days after NEUG administration. Both events
resolved without sequeale.
[0290] Thirty one of the 41 NEUG-treated subjects experienced at least 1 adverse event.
The incidence of AEs among NEUG - and pegfilgrastim-treated subjects was comparable
(75.6% and 70% respectively).
[0291] A summary of commonly reported adverse events (AEs greater than or equal to 5%
of all subjects) for Part B is provided in Table 17.



[0292] The most commonly reported adverse event considered related to NEUG was bone
pain, a typical adverse reaction associated with all G-CSF products, which was reported in 5
patients 4 listed in the table above, plus one Part A subject receiving 450 µg/kg). In all cases,
the bone pain was NCI-CTCAE grade 1 -2 in intensity, transient in duration and resolved
without sequelae. Grade 1 elevations in alkaline phosphatase and uric acid occurred
following administration of NEUG in Cycle 0; these events were deemed to be not clinically
significant by the Investigators and resolved without intervention. These are expected effects
in patients receiving a G-CSF (e.g., Neulasta®).
[0293] Other commonly reported adverse events during chemotherapy cycles (nausea,
vomiting, alopecia, stomatitis) were consistent with anticipated adverse events in patients
receiving the doxorubicin/docetaxel regimen.
[0294] The majority of reported AEs were of NCI CTC Grade 1 or 2 severity. Four AEs
were reported as serious adverse events. Two subjects, one receiving 150 µg/kg and one 450
µg/kg, experienced vomiting that caused hospitalization and one of these subjects
experienced a second SAE in the following chemotherapy cycle; vomiting that was mild in
intensity but caused or prolonged hospitalization. A third subject received 450 µg/kg was
hospitalized for febrile neutropenia. The events were considered unrelated to NEUG.
e. Pharmacokinetics
[0295] All subjects receiving NEUG were sampled for serum NEUG concentrations over
the course of the study. The drug was detected using a sandwich enzyme-linked
immunosorbent assay (ELISA) specific for NEUG. The serum drug concentration-time data
was subjected to PK analysis using WinNonlin Enterprise Edition, Version 4.1 or higher,
using noncompartmental or model-based analysis.

[0296] The following PK parameters were obtained: area under the curve (AUC),
clearance (CL/F), volume of distribution (Vz/F), maximum concentration (Cmax), absorption
half-life (t1/2, abs), elimination half-life (t1/2, elim), and mean residence time (MRT).
Pharmacokinetic data were assessed for linearity across the dose range employed in the
protocol.
[0297] Pharmacokinetic parameters from cycle 0 (pre-chemotherapy) are summarized in

[0298] Drug exposure as measured by maximum serum NEUG concentration and area
under the time-concentration curve increased in a dose-dependent mariner. Serum
concentrations for subjects in the initial 50 µg/kg dose cohort were consistently below the
lower limit of quantization (6.3 ng/mL). Tmax was in the range of 6-24 hours for all doses
from 150 through 450 µg/kg. Cmax ranged from 72.7 ± 59.7 (mean ± SD) ng/mL at a dose of
150 µg/kg to 294 ±351 ng/mL, at a dose of 450 µg/kg. Correspondingly, AUC ranged
from 1758 ± 1675 ng/mL*hr at a dose of 150 mcg/kg to 10131 ± 9563 ng/mL*hr at a dose of
450 µg/kg. Cycle 1 ranges were similar. The mean elimination half-life of NEUG ranged
from 14-30 hours.
[0299] As noted in "Study Characteristics" (section 4, above), subjects in Part A received
the first dose of NEUG at least 2 weeks prior to the start of chemotherapy (cycle 0) for an
initial assessment of safety and effects on absolute neutrophil count ("ANC") in the absence
of cytotoxic chemotherapy. After a minimum of 2 weeks follow-up, subjects received NEUG
at the same dose following chemotherapy in cycles 1 and 2 if there were no dose-limiting

adverse events considered related to NEUG in cycle 0 and the subject continued to meet all
eligibility criteria. NEUG was administered 24 hours following chemotherapy
administration. FIG. 24A and 24B show the ANC and WBC count for subjects that received
NEUG during cycles 1 and 2.
f. Pharmacodynamics and establishment of Part B dosages
[0300] Analysis of the data from Part A of Phase I of the study yielded the following
observations:
1. NEUG induces a dose-dependent rise in WBC and ANC rise in Cycle 0 (prior
to chemotherapy) (see cycle 0 data at FIG. 24A and B).
2. ANC increases in Cycle 0 were comparable to historical data for pegfilgrastim
at equimolar doses
3. As anticipated, WBC and ANC drop following chemotherapy
4. Recovery from Nadir ANC appears dose related
5. ANC and WBC returned to normal by day 15
[0301] Based on these observations and demonstration of safety at all dose levels in Part A,
the doses chosen for the Part B evaluation were 300 and 450 µg/kg. As described above,
subjects were randomized to NEUG 300 M-g/kg, NEUG 450 µg/kg, or pegfilgrastim at the
approved fixed dose of 6 mg. Subjects received the NEUG or pegfilgrastim one day
following doxorubicin/docetaxel (administered for 2 cycles, 21 days apart). Data for Part B
includes the cycle 1 ANC profiles of the population. Results are summarized in Figure 28
and Table 18, below.
[0302] The incidence of grade 3 and 4 neutropenia, and the ANC profiles during Cycle 1
were determined in 48 of 51 treated subjects as show in Table 19. Note that 70-80% of
patients treated with doxorubicin/docetaxel get Grade 4 neutropenia with durations average
of 5 days in the absence of prophylactic G-CSF treatment.



[0303] Mean ANC curves for the treatment groups are presented in FIG. 28.
[0304] NEUG is effective for treating grade 3, grade 4 and febrile neutropenia. In the
absence of G-CSF treatment for this chemotherapy regimen, the incidence of febrile
neutropenia is about 40%, A dose-related elevation in ANC and a lower rate of neutropenia
than is expected with doxorubicin/docetaxel were observed following administration of
NEUG, There were no unexpected or serious adverse events attributed to NEUG.
[0305] The incidence of grade 3 and grade 4 neutropenia was higher in patients receiving
300 µg/kg NEUG than those receiving pegfilgrastim (Neulasta®) and the rate of return to
normal ANC also appeared slower in patients who received 300 µg/kg NEUG than in those
subjects who received pegfilgrastim. The ANC profiles in patients who received NEUG at
450µg/kg and those who received pegfilgrastim were similar, though the ANCs during
recovery from neutropenia were generally lower in patients who received NEUG than in
patients receiving pegfilgrastim. In summary, NEUG at these doses appears to provide
similar effect as pegfilgrastim.
f. PK/PD profile, Phase I, Part B
[0306] The PK/PD profile from patients receiving 450 µg/kg NEUG one day after
doxorubicin/docetaxel administration in cycle 1 of treatment for breast cancer is shown in
FIG. 29. Cmax for NEUG is achieved within one day of administration and gradually falls to
undetectable levels by day 10. Following administration of NEUG, the ANC rises to a peak
by day 4 and then, as expected in patients receiving doxorubicin/docetaxel and G-CSF
treatment, the ANCs fall to a nadir on day 8 and return to normal on day 10. By day 12,
ANC values are in the normal range and NEUG is undetectable. Note that in patients who do
not receive prophylactic G-CSF treatment, the duration of nadir ANC and time to reach
recovery ANC are much longer (e.g., 5-7 days). After a dose of 450 µg/kg, the NEUG

median elimination half-life was approximately 30 hours, as compared to the 15-80 hours
reported for a standard dose of pegfilgrastim..
g. Additional differences between NEUG and pegfilgrastim
[0307] More detail of the differences between NEUG and pegfilgrastim at the tested doses
in effectiveness in hastening the recovery form neutropenia is evident in comparison of the
individuals ANC profiles in cycle 1 of treatment. The peak ANCs in all groups were very
similar, nadir ANCs in subjects receiving NEUG at 300 µg/kg were lower than in subjects
receiving NEUG at 450 µg/kg, and the ANC nadirs in subjects receiving pegfilgrastim were
on average the highest. Recovery from nadir ANC to baseline occurred by day 14 in all
treatment groups, but was slower for those receiving 300 µg/kg NEUG, than 450 µg/kg
NEUG, and most rapid for subjects receiving pegfilgrastim.
[0308] Available published data for a pegfilgrastim trial with a similar prechemotherapy
administration were compared to NEUG PK/PD data from patients who completed the Phase
I through the scheduled cycle 0 (pre-chemotherapy). Results of this comparison were as
follows:
1. Emax (maximum observed ANCs) at NEUG dose of 150 µg/kg matches the
30 µg/kg dose of pegfilgrastim in Cycle 0, a dose later demonstrated to be
inferior for efficacy to the confirmed efficacious pegfilgrastim dose of 100
Jig/kg.
2. Emax for 300 and 450 µg/kg Neugranin doses are more consistent with Cycle
0 levels for 100 µg/kg dose of pegfilgrastim.
3. At 300 and 450 µg/kg NEUG median Cmax and median Emax are nearly the
same, thus Cmax continued to predict Emax.
4. ANC increases were comparable to published data for pegfilgrastim at
equimolar doses.
[0309] As discussed above, PK/PD assessment in animals and in man was consistent with
an estimate of NEUG and pegfilgrastim dose equivalence when dosed on an equimolar basis.
In mice, equivalent AUCANC were achieved with a 7.7 fold higher dose thane pegfilgrastim.
Because albumin contributes significantly to the molecular weight of NEUG, and Neulasta®
(pegfilgrastim) is dosed base on the weight of the rhG-CSF (not including the contribution of

the polyethylene glycol in pegfilgrastim), a 4.5 fold greater dose of NEUG (based on weight)
is predicted to be as effective as an equal dose of Neulasta® (pegfilgrastim). Efficacy data in
animals were consistent with a 4.5-7.7 fold equivalence to pegfilgrastim (1 mg pegfilgrastim
= 4.5-7.7 NEUG). Non-clinical safety and effect data are consistent with this dose estimate
and when considered with available clinical data, form the basis for the doses elected for
clinical evaluation.
h. Results of Phase I
[0310] The Results from the Phase I pharmacokinetic evaluation are as follows.
NEUG was detected in serum samples from all subjects treated with NEUG at doses
of 150 µg/kg, 300 µg/kg and 450 µg/kg on Cycle 0 and Cycle 1.
In Cycle 1, NEUG was detected uptown 144 hours in most subjects (45/50 sampled)
in the 150 mg/kg, 300 µg/kg and 450 µg/kg dose groups. Virtually no cycle to cycle
drug accumulation was observed.
Drug exposure was higher in Cycle 1 and in Cycle 0 (pre-chemotherapy) with each
dose group. The increased exposure to NEUG in Cycle 1 is likely due to the
decreased number of neutrophils, which plan a role in the receptor-mediated clearance
of G-CSF.
The median elimination half-life of NEUG in Cycle 1 was about 36 hours for dose
group 300 µg/kg and 30 hour for dose group 450 µg/kg. The elimination half-hfe is
reported to be 3-4 hours for filgrastim and 42-67.5 hours, depending on dose, for
pegfilgrastim.
Statistically significant differences across doses were observed in the time to maximal
serum concentration (tmax) and the absorption half-life (t1/2,abs)- Both of these
parameters increased with increasing NEUG dose. No other dose normalized PK
parameters showed statistically significant differences across doses.
Example 11: PHASE II
[0311] Phase II of the study was a controlled, randomized trial, conducted in 334 subjects
with breast cancer who received up to 4 doses of doxorubicin/docetaxel. The study, was
conducted at approximately 50 clinical sites, and consisted of a two-way randomized pilot

phase to assess the safety and effect of subeutaneously administered NEUG versus
pegfilgrastim, followed by a main phase in which subjects were randomized to pegfilgrastirn
and two, well-tolerated doses of NEUG (1:1:1) selected based on the pilot phase. The sample
size for the main phase was powered to establish non-inferiority of NEUG to pegfilgrastim
with regard to the primary endpoint, duration of sever (grade 4) neutropenia (DSN) during
chemotherapy cycle 1. The study design is shown schematically below.

1. Objectives
[0312] The primary objectives of Phase II were to select doses of NEUG demonstrating a
comparable effect to pegfilgrastim and to assess the duration of severe neutropenia (DSN) in
cycle 1 of chemotherapy after treatment with NEUG. Secondary objectives were to assess the
DSN in cycles 2-4, to assess the time to absolute neutrophil count recovery and rates of

febrile entroopenia in cycles 1-4; and to assess the safety, tolerability, pharmacokinetics (in
cycle 1), and immunogenicity of NEUG.
2. Patient characteristics
[0313] For Phase II, patients were screened based on the following characteristics or
parameters:
[0314] Inclusion;
1. Patients with histologically-confirmed breast cancer scheduled to receive
doxorubicin 60 mg/m2 and docetaxel 75 mg/m2
2. 18 years of age or older
3. Adequate hematologic function:
4. ANC>1500/mm3
5. Platelets > 100,000/mm3
6. Adequate hepatic and renal function:
7. Serum creatinine < 1.5 x upper limit normal
8. Total bilirubin within normal limits (WNL) for local laboratory
9. Serum transaminases (SGOT/SGPT) < 1.5 x upper limit normal
10. Alkaline phosphatase < 2.5 x upper limit normal
11. Eastern Cooperative Oncology Group ("ECOG") performance status 0-2
12. Eligible to receive doxorubicin based on a left ventricular ejection fraction
(LVEF) within normal limits
13. Have the ability to understand the requirements of the study, provide written
informed consent (including consent for use and disclosure of research-related
health information) and comply with the study protocol procedures.
[03151 Exclusion:
L More than 1 prior chemotherapy regimen (including adjuvant therapy if given
within the last 12 months)
2. A cumulative anthraeyelme dose that would preclude 4 full-dose cycles of
doxorubicin in this study
3. Prior chemotherapy/immunotherapy within 30 days prior of study
chemotherapy (within 6 weeks of study chemotherapy for nitrosoureas (BCNU,

CCNU) or mitomycin-C)
4. Concomitant trastuzumab (Herceptin)
5. Received any investigational agent in the past 30 days
6. Cardiac history, signs or symptoms that, in the Investigator's opinion, preclude
the use of an anthracycline-based chemotherapy regimen
7. Prior surgery within 2 weeks of study chemotherapy
8. Prior radiation therapy within 4 weeks of study chemotherapy (except spot
irradiation for bone metastases)
9. Prior high-dose chemotherapy with hematopoietic stem cell transplant
10. Prior use of G-CSF, GM-CSF or erythropoietin within 4 weeks of study
chemotherapy
11. Received systemic antibiotics within 72 hours of study chemotherapy
12. History of myeloid malignancy or myelodysplasia

13. Known brain metastases unless adequately treated (surgery or radiotherapy),
no evidence of progression with a minimum of 3 weeks observation and
neuroiogicatly stable off anticonvulsants and steroids.
14. Known sickle cell disease
15. Diagnosis of adult respiratory distress syndrome (ARDS)
16. Known history of allergies to yeast-derived products
17. Known hypersensitivity to E coli-derived proteins, pegfilgrastim, filgrastim,
or any other component of pegfilgrastim
18. Pregnant female or nursing mother. (All females with an intact uterus must
have a negative serum pregnancy test at screening. All non-sterile or non-
postmenopausal females must practice a medically accepted method of
contraception over the course of the study and for 30 days after the last dose of
study agent.)
19. Males who do not agree to use effective contraception throughout the study
and for a period of 30 days after the last dose of study agent
20. Known HIV positive or active hepatitis (Patients with unknown status will not
be tested)
[0316] Subjects were removed from further treatment for the following reasons:

1. Disease progression
2. Unacceptable toxicities despite optimal treatment
3. Intercurrent illness at the investigator's discretion
4. Doxorubicin regimen - Maximum lifetime permissible cumulative dose
reached (see eligibility criteria)
5. Withdrawal of consent
6. Non-compliance/Loss to follow-up
7. Pregnancy
[0317] If treatment with study drug was stopped, subjects remained on study were followed
at least 30 days following the final dose of any study drug for scheduled safety and PK
assessments.
3. Study agent
[0318] NEUG (recombinant human albumin- human granulocyte colony stimulating factor,
rHSA-GCSF), is a fusion protein with a molecular mass of approximately 85kDa connected
in a single chain comprising residues 1-585 corresponding to the mature form of HSA and
residues 586-759 corresponding to the mature form of human G-CSF. The therapeutic
moiety of NEUG is recombinant human DNA-derived G-CSF.
[0319] NEUG was supplied as a sterile, lyophilized formulation in single-use Type 1 glass
vials and stored at 2-8°C. Upon reconstitution with 1.0 ml of sterile water for injection, each
vial contains 50 mg/ml (50 mg/vial deliverable) NEUG in 20 mM sodium phosphate, 180
mM, mannitol, 60mM trehalose dehydrate, 0.01% (w/v) polysorbate 80, pH 6.0.
[0320] The composition of the drug product used in Phase II is shown in Table 11.
Difference between the NEUG formulations used in Phase I and Phase II are shown below in
Table 20.


■ ■■■•■■
[0321] The formulation used in Phase I was quite stable, with a shelf-life of at least 2 years.
Studies demonstrated that higher ionic strength and lower pH further stabilized the API at
higher concentration (> 25 mg/mL) (data not shown). To this end, the Phase II formulation
has a lower pH (6.0 vs 7,2) and higher phosphate concentration (20 vs. 10 mM), Forced
degradation studies demonstrate that this formulation protects the drug substance in the liquid
state from vigorous shaking, repeated freeze-thawing, and concentration induced aggregation.
Freeze drying of the Phase II formulation also produces well-formed cakes.
[0322] Commercially available Neulasta® (pegfilgrastim) is supplied in 0.6 ml prefilled
syringes for subcutaneous injection. Each syringe contains 6 mg pegfilgrastim (based on
protein weight), in a sterile, clear, colorless, preservative-free solution (pH 4.0) containing
acetate (0.35 mg), sorbitol (30.0 mg), polysorbate 20 (0.02 mg), sodium (0.102 mg) in water
for injection. USP.
[0323] NEUG (30,40,50, or 60 mg) or Neulasta® (pegfilgrastim) (6 mg) was administered
by subcutaneous administration.
Dose Rationale
[0324] The data from Phase I demonstrated that doses of NEUG of 300 and 450 µg/kg were
safe and well tolerated. Moreover, compared to the approved fixed doses of pegfilgrastim,
both doses of NEUG resulted in similar effects on ANC profiles in breast cancer patients
receiving cytotoxic chemotherapy. The AUC for the ANC profiles serves as a single-point
measure of effect. There was no statistically significant difference among these treatment

groups in terms of AUCANC, however, the AUC for the 450 µg/kg group is slightly higher
than that observed for the 300 µg/kg group and nearly identical to that observed for the
pegfilgrastim group (FIG. 30). Based on available data, it was estimated that 300 µg/kg
NEUG was less effective than pegfilgrastim and 450 µg/kg approximates a minimum
necessary dose to provide equivalent effect to pegfilgrastim.
[0325] The intent of a fixed dose is to identify doses that will provide patients wit a dose
sufficient to provide efficacy and safety regardless of patient weight. Based o the results of
Phase I, it as estimated that 450 µg/kg NEUG may be a minimum dose necessary to provide
similar effect as pegfilgrastim, and > 300 µg/kg was set as the minimum dose for further
evaluation in Phase II. To select fixed doses of NEUG, the patient population (breast cancer)
for Phase II was modeled. Using 40-100 kg weight range, a 30 mg fixed dose provides the
heaviest patient with a minimum dose (300 µg/kg or 0.3 mg/kg), while approximately 75% of
patients receive at least the target dose, 450 mg/kg, at a fixed dose of 40 mg. Thus, the doses
selected for evaluation in Phase II were 30 mg, 40 mg and 50 mg. These provide an average
70 kg patient with 0.42,0.57 and 0.71 mg/kg doses, respectively.
[0326] The equivalent dose per kilogram based on the fixed doses evaluated in this trial is
provided in Table 21.

[0327] The nonclinical safety for NEUG provides additional support for the expectation of
safety at these doses. Exposure in patients at these fixed doses (AUC and Cmax) is expected
to be lower than exposure at well tolerated doses in monkeys. For example, Cmax and AUC
in the monkey at the well-tolerated dose of 1 mg/kg was 12-fold higher than exposure in
patients at 0.45 mg/kg suggesting a further margin of safety exists for higher dose evaluation

in patients and in a repeat-dose toxicology study in monkey, doses up to and including 10
mg/kg were well tolerated. Doses of pegfilgrastim as high as 0.3 mg/kg have been
demonstrated to be safe in patients.
4. Study characteristics
a. Study Schedule and Duration
[0328] This study was a controlled, randomized trial conducted in approximately 330
subjects with breast cancer scheduled to receive up to 4 doses of doxorubicin/docetaxel. The
study, which was conducted at approximately 45 clinical sites, consisted of two phases, a
pilot phase and a main phase.
[0329] The pilot phase, Part A, consisted of a two-way randomized study to assess the
safety and effect of NEUG versus pegfilgrastim, with sequential enrollment to the following
doses: NEUG 30 mg (N=10) vs. pegfilgrastim (N=5); NEUG 40 mg (N=20) vs. pegfilgrastim
(N=10), and NEUG 50 mg (N=20) vs. pegfilgrastim (N=10). In a further study, NEUG 60
mg (N=20) vs. pegfilgrastim (N=10) could also be tested. In the Part A pilot phase, subjects
were randomized in a 2:1 ratio of NEUG to pegfilgrastim with a total of 10 subjects in the 30
mg cohort and 20 subjects for each of the other cohorts. NEUG or pegfilgrastim was
administered to subjects 24 hours after the chemotherapy treatment in each cycle. Subjects
were assigned to treatment groups using a stratified randomization for balance among
treatment groups based on weigh (<50 kg, > 50 kg and < 80 kg, or > 80 kg), prior
chemotherapy exposure and global location.
[0330] Following the pilot phase, 255 subjects were randomized (1:1:1) to pegfilgrastim
and the two well tolerated doses of NEUG with the more comparable effect to pegfilgrastim
in the pilot phase (a 3-arm, balanced parallel-randomized phase). NEUG or pegfilgrastim
was administered 24 hours after the chemotherapy treatments in each cycle. Subjects were
assigned to treatment groups using a stratified randomization for balance among treatment
groups based on weight (<50 kg, > 50 kg and < 80kg, or >80 kg).
[0331 ] During the pilot phase, adverse events were reviewed on an ongoing basis.
Escalation of the dose from 30 through 50 mg occurs unless the ongoing review of data
suggested a safety concern. If the Cycle 1 ANC profile for Neugranin at 40 mg appeared
inferior to the profile observed from pegfilgrastim patients and 50 mg of Neugranin is safe,

then an additional arm may be randomized in a 2:1 ratio of Neugranin at 60 mg to
pegfilgrastim with a total of 30 patients in the cohort,
[0332] Each dose level of NEUG is compared to pegfilgrastim for safety and efficacy.
Table 22 summarize the patient allocation for Phase II, Part A and Part B,

Safety Evaluation:
[0333] The safety of NEUG was assessed by evaluation of the type, frequency, and severity
of AEs, changes in clinical laboratory tests (hematology and clinical chemistry),
immunogenicity, physical examinations, and the monitoring of vital signs over time. All AEs
and laboratory toxicities were graded based on the National Cancer Institute Common
Terminology Criteria for Adverse Events (NCI-CTCAE Version 3.0,12 December 2003).
[0334] Adverse events were captured from the start of study drug administration through 30
days following the final dose of any study drug. Serious adverse events (SAE) were captured
from the time of consent through 30 days following the final dose of any study drug.
Laboratory assessments were obtained as outlined in the Schedule of Assessments.
c. Concomitant therapy
Chemotherapy
[0335] The chemotherapy regimen for this trial consisted of doxorubicin 60 mg/m2 and
docetaxel 75 mg/m2 administered sequentially by intravenous infusion on day 1 of treatment
for up to four 21-day cycles.
[0336] Prior to receiving each cycle of therapy, subjects were required to have an absolute
neutrophil count (ANC) > 1000/mm3 and platelets > 100,000/rnm3. Treatment could be

delayed up to two weeks for hematologic recovery. A 25% dose reduction of chemotherapy
doses was allowed for grade 3-4 non-hematologic toxicities, two grade 3-4 infectious
episodes, or grade 4 thrombocytopenia. The use of prophylactic antibiotics or other
hematopoietic growth factors was prohibited during trial participation.
[0337] The combination of doxorubicin and docetaxel has been reported to have significant
clinical activity in patients with breast cancer. However, the combination is highly
myelosuppressive with higher rates of grade 3 or 4 neutropenia than other standard regimens.
[0338] Even with the addition of CSFs, the combination of doxorubicin and docetaxel has
induced Grade 4 neutropenia in 79% of patients and febrile neutropenia rates of 9-18%. This
doxorubicin/docetaxel regimen has been used in studies of new agents to prevent neutropenia
and its complications. Therefore, the combination of doxorubicin and docetaxel is an
appropriate chemotherapy regimen to study the potential of a new agent like NEUG.
Doxorubicin
Pharmacologic Data
[0339] Doxorubicin hydrochloride is an anthracycline antibiotic obtained from
streptomyces peucetius var caesius which binds directly to DNA base pairs (intercalates) and
inhibits DNA and DMA-dependent RNA synthesis, as well as protein synthesis. Doxorubicin
is active in ail phases of the cell cycle but maximally cytotoxic in S phase. Excretion of the
drug is predominately by the liver; renal clearance is minor.
Pharmaceutical Data
[0340] The drug is marketed commercially in 10,20 50,100 or 200 mg vials. Lyophilized
preparations may be reconstituted with sterile water for injection, dextrose 5% solution, or
0.9% saline for injection.
Side Effects and Toxicity
[0341] Myelosuppression, primarily leukopenia, with a nadir of approximately 10-14 days,
and cardiotoxicity, including a rare, acute pericarditis-myocarditis syndrome and a delayed,
cumulative dose related cardiomyopathy are the dose-limiting toxicities of doxorubicin.
[0342] Marked alopecia and moderate nausea/vomiting are expected toxicities.
Extravasation reactions producing local skin and tissue damage at the site of inadvertent
extravasation, stomatitis, hyperpigmentation of the skin (particularly the nailbeds), and a
"recall" phenomenon at sites of previous irradiation have been reported.

Docetaxel
Pharmacologic Data
[0343] Docetaxel is a semisynthetic taxoid that binds to free tubulin and promotes assembly
of stable microtubules, interfering with mitosis and cell replication (cell cycle specific for M
phase). Docetaxel is extensively protein-bound, extensively metabolized in the liver, with
fecal excretion of approximately 75% of the dose within 7 days.
Pharmaceutical Data
[0344] Docetaxel (Taxotere™, Sanofi Aventis) is provided in 80mg/2 mL or 20 mg/0.5 ml
single-dose vials with an accompanying diluent (13% ethanol in Water for Injection) vial.
Each ml of Taxotere contains 40 mg of docetaxel (anhydrous) and 1080 mg polysorbate 80.
Side Effects and Toxicity
[0345] Docetaxel should not be given to patients who have a history of severe
hypersensitivity reactions to docetaxel or other drugs formulated with polysorbate 80 such as
etoposide and vitamin E.
[0346] Patients who experience severe hypersensitivity reactions should not be
rechallenged. All patients receiving docetaxel should be premedicated with corticosteroids as
outlined below.
[0347] Mild to moderate liver impairment results in delayed metabolism by 27% and a 38%
increase in systemic exposure (AUC). Docetaxel should not be given to patients with SGOT
and/or SGPT > 1.5 times normal limits and alkaline phosphatase > 2.5 times normal limits.
Fluid retention occurred in 17% (moderate) and 6% (severe retention) of patients in phase III
studies despite corticosteroid premedication. Severe neurosensory symptoms (paresthesia,
dyesthesia, pain) have been observed.
[0348] Expected side effects include myelosuppression, primarily leukopenia, with a nadir
of approximately 9 days with recovery by day 15-21. Alopecia, nail and cutaneous changes,
stomatitis, myalgia/arthralgia, nausea/vomiting, and hypotension have been reported.
Chemotherapy Dosage. Administration and Dose Modifications
[0349] On day 1 of each treatment cycle, chemotherapy (doxorubicin followed by
docetaxel) was to be administered.

[0350] Doxorubicin was administered at a dose of 60 mg/m by IV bolus through the side
arm of an infusing intravenous line or central venous catheter to avoid extravasation injury.
[0351] Docetaxel 75 mg/m2 was diluted in 250 ml 0.9% saline or 5% dextrose solution and
administered intravenously over approximately 1 hour via a polyethylene-lined infusion set.
Vital signs were obtained immediately prior to and after the end of the docetaxel infusion.
[0352] Subjects experiencing severe hypersensitivity reactions or non-hematologic
toxicities that preclude further cycles of chemotherapy were to be removed from study
treatment and complete follow-up.
Chemotherapy Pre-medication
[0353] Oral (IV as needed) corticosteroids (such as dexamethasone 8mg BID) was
administered for three days starting 1 day prior to docetaxel administration in order to reduce
the incidence and severity of fluid retention and hypersensitivity reactions.
[0354] The use and selection of anti-emetic agents or other pre-medications (e.g. H2
antagonists) was left to the discretion of the treating physician.
Prohibited Medications
[0355] Subjects were not to receive any of the following medications and or procedures
during this study and for the additional times specified below:
1. Systemic antibiotics within 72 hours of cycle 1 chemotherapy.
2. Other investigational agents within 30 days of initiating study agent and for
the duration of the trial
3. Subsequent cycles of chemotherapy should not be initiated until 14 days
following dosing with NEUG.
4. Cytokines, other hematopoietic growth factors and prophylactic antibiotics for
the duration of the trial unless prolonged or febrile neutropenia occurs. If the
subject is treated with G-CSF at any time between the screening period and
Day 0 they will not be eligible to receive NEUG and will be discontinued from
the study.
Allowed Medications
[0356] Subjects were allowed to continue their baseline medications(s). The daily dose of
each medication was maintained throughout the study if possible. If for any reason deemed
necessary by the investigator, a subject required additional medication(s) or change of dose,

the medication(s), route of administration, and the indication for which it was given was be
recorded.
[0357] Subjects experiencing severe hypersensitivity reactions or non-hematologic
toxicities that preclude further cycles of chemotherapy were removed from study treatment
and completed follow-up.
d. Pharmacokinetics
[0358] All subjects receiving NEUG were sampled for serum NEUG concentrations during
cycle 1. The drug was detected using a sandwich enzyme-linked immunosorbent assay
(ELISA) specific for NEUG. The serum drug concentration-time data was subjected to PK
analysis using WinNonlin Enterprise Edition, Version 5.0 or higher, using noncompartmental
or model-based analysis. The following PK parameters were determined: area under the
curve (AUC), clearance (CL/F), volume of distribution (Vz/F), maximum concentration
(Cmax), absorption half-life (tl/2, abs), elimination half-life (t1/2, elim), and mean residence
time (MRT).
e. Immunogenicity
[0359] Serum samples for antibodies to NEUG were obtained prior to dosing on Day 1 of
every NEUG cycle and at the end of treatment visit (approximately 30 days after the last
dose) in subjects receiving NEUG. If at any time during the study a subject developed a
positive anti-NEUG antibody response, a repeat sample was obtained approximately 6
months after the final NEUG dose; if this sample was positive, a sample was obtained at 12
months. The protocol was later amended to require 6 and 12 month immunogenicity samples
from all subjects.
5. Results
a. general
Statistical Methods
[0360] The sample size of about 85 subjects per arm in the main phase of this trial (Part B)
was chosen to provide 91% power to establish non-inferiority of NEUG to pegfilgrastim with
regard to the primary endpoint of mean duration of severe neutropenia (DSN) in cycle 1, with
a non-inferiority margin of 1 day and an overall 1-sided significance level adjusted for
multiple testing (by the Hochberg method) of 0.025. Sample sizes were calculated based on

the normal approximation for two independent groups, an estimate of 1.6 days as the within-
treatment standard deviation of cycle 1 DSN, and a maximum rate of 20% not evaluable for
the primary endpoint of cycle 1 DSN.
[0361] Efficacy comparison was made between the two selected NEUG doses (either 40 mg
and 50 mg) and pegfilgrastim, based on subjects in the 3-arm randomized phase (Part A).
[0362] Secondary efficacy analyses include the DSN in each of chemotherapy cycles 2
through 4, depth of ANC nadir in each of the cycles 1 through 4, rates of FN (defined as
ANC < 0.5 x 109/L with coincidental oral equivalent temperature > 38.2°C) by cycle and
across all cycles, and times to ANC recovery to >1.5 x 109/L in all cycles.
[0363] The data related to secondary efficacy analysis was analyzed using appropriate
statistical methods. Safety, PK, and immunogenicity parameters were analyzed by
descriptive statistical methods.
[0364] For frequency and severity of adverse events, and for laboratory toxicity grading,
counts and rates are presented.
Efficacy Measures
[0365] Complete blood counts ("CBC") were obtained on day 1, 3 and daily from day 5
until ANC > 2.0 x 10 /L after the nadir, then twice weekly, and at the end of treatment.
b. Efficacy of Phase II, Part A
[0366] Of the 78 subjects enrolled in the pilot phase of the study, 13 subjects did not
complete the study, 3 (27.3%) treated with NEUG 30 mg, 3 (14.3%) treated with NEUG 40
mg, 3 (15.0%) treated with NEUG 50 mg, and 4 (15.4%) treated with pegfilgrastim. The
most frequent reasons for early discontinuation were withdrawal of consent (7 subjects) and
decision of the investigator (3 subjects). One NEUG 30 mg subject was withdrawn due to an
adverse event (diabetic foot).
[0367] The incidence of sever neutropenia and the mean duration of severe neutropenia
(DSN) were similar across treatment groups in each chemotherapy cycle; however, the time
to ANC recovery and the incidence of febrile neutropenia suggested that NEUG 30 mg was
not quite as effective as NEUG 40 mg, NEUG 50 mg, or pegfilgrastim.
[0368] During Cycle 1, the proportion of subjects experiencing febrile neutropenia was
20.0%, 9.5%, 10.0% and 8.0% for the NEUG 30 mg, 40 mg, 50 mg and pegfilgrastim group,

respectively. Febrile neutropenia was observed for only three additional subjects during
Cycles 2-4, one each in the NEUG 30 mg, NEUG 40 mg and pegfilgrastim groups. FIG. 5
shows the ANC profile of a subset of patients receiving either NEUG 30 or pegfilgrastim and
who later presented with grade 4 neutropenia.
[0369] In Cycle 1, the mean DSN was similar for NEUG 30 mg (0.9 days), NEUG 50 mg
(1.1 days), and pegfilgrastim (0.9 days). Although the mean DSN was slightly longer for
NEUG 40 mg (1.6 days) than the other three treatments, the differences among treatments
were all less than 1 day, the criterion to consider the treatments equivalent in the main phase.
The median DSN was 0 or 1 day in all four treatment groups.
[0370] Summary statistics for the incidence and duration of Grade 3 or 4 neutropenia
followed a similar pattern, i.e., the NEUG 30 mg, NEUG 50 mg, and pegfilgrastim groups
had similar outcomes, while the incidence and duration of Grade 3 or 4 neutropenia were
slightly higher for the NEUG 40 mg group than for the other treatment groups. The number
of subjects in the pilot phase (Part A) was fairly small, and the observed differences were not
statistically significant. NEUG 40 mg and NEUG 50 mg were selected for further evaluation
in Part B, the 3-arm randomized phase of the study.
c. Efficacy of Phase II, Part B
[0371] Of the 256 subjects enrolled in the main phase of the study, 18 subjects did not
complete the study; 10(11.6%) treated with NEUG 40 mg, 5 (6.0%) treated with NEUG 50
mg, and 3 (3.5%) treated with pegfilgrastim. The most frequent reasons for early
discontinuation were withdrawal of consent (7 subjects) and AEs (4 subjects), including 2
deaths. The investigator considered all of these AEs to be not related to study medication or
chemotherapy. In the main phase, 1 (1.2%) NEUG 40 mg subject was withdrawn before
being treated with study drug.
[0372] The incidence and duration of sever neutropenia in Cycle 1 are summarized in
Table 23.



[0373] The incidence of severe neutropenia ranged from 58.1% in the pegfilgrastim group
to 65.5% in the NEUG 50 mg group. The treatment effect was not statistically significant
(p=0.559). The treatment groups were comparable for Cycle 1 DSN, with mean values of
1.0, 1.3, and 1.2 days for the NEUG 40 mg, NEUG 50 mg, and pegfilgrastim groups,
respectively. The 95% and 97.5% two-sided confidence intervals for differences between
NEUG and pegfilgrastim were strictly less than 1 day for both NEUG doses. This analysis
established non-inferiority of NEUG to pegfilgrastim. Across treatment cycles, the
incidences of severe neutropenia and Grade 3 or 4 neutropenia were lower in Cycles 2-4
than in Cycle 1. The mean DSN and mean duration of Grade 3 or 4 neutropenia were smaller
in Cycles 2-4 than in Cycle 1. Within treatment cycles, the treatments were similar, and
treatment effect was not significantly different for any of these parameters in any
chemotherapy cycle.
[0374] The DSN were compared in patients grouped into weight quartiles to determine if
the fixed doses of NEUG provided adequate support for patients of all weights. These results
show that all weight groups were adequately supported, as there is no significant difference in
the mean DSN among weight subgroups (Table 24).


[0375] Febrile neutropenia is summarized for all cycles in Table 25. During Cycle 1, the
proportion of subjects experiencing febrile neutropenia was 2 subjects (3.5%), 5 subjects
(6.0%), and 2 subjects (2.3%) in the NEUG 40 mg, NEUG 50 mg, and pegfilgrastim groups,
respectively. Febrile neutropenia was observed for only three additional subjects during
Cycles 2-4, 2 subjects in the NEUG 40 mg group and 1 subject in the pegfilgrastim group.
The treatment effect was not statistically significant in any chemotherapy cycle.

[0376] There were no significant differences between treatments for duration of sever
neutropenia in cycles 2-4 (Table 26).


[0377) The mean time to ANC recovery (> 1.5 x 109/L) was 2.0, 2.1, and 2.6 days for the
Neugranin 40 mg, NEUG 50 mg, and pegfilgrastim groups, respectively (Table 27). There
were no significant differences between treatment groups for the depth of ANC nadir or time
to nadir.


[0378] Serum Neugranin concentrations were determined using a validated sandwich
ELISA with a lower limit of quantification (LLQ) of 6.312 ng/mL. Pharmacokinetic
parameters were calculated using noncompartmental modeling techniques, with the exception
of the absorption half-life, which was determined using a first-order absorption, first-order
elimination one-compartment model. Modeling was performed with WinNonlin Professional
(version 5.0.1). Serum NEUG concentrations were determined in chemotherapy Cycle 1 in
all subjects treated with NEUG in Phase II. In the Part A of Phase II, the median elimination
half-life of NEUG was 33 hours in the 30 mg dose group, 46 hours in the 40 mg dose group,
and 18 hours in the 50 mg dose group (Table 28). In Part B, the median elimination half-life
of NEUG was 40 hours for 40 mg dose group, and 39 hours for the 50 mg dose group (Table
29). During the Part A, PK sampling was more frequent (pre-dose, 3h, 6h, 12h, 24h Day 3,
Day 5-9, Day 11) than for Part B (pre-dose, Day 3, Day 5-8).

[0379] Serum pegfilgrastim concentrations were determined using a validated sandwich
ELISA in chemotherapy Cycle 1 in all subjects treated with pegfilgrastim in Phase n. In Part
A, the median elimination half-life of pegfilgrastim was about 40 hours. In Part B, the
median elimination half-life of pegfilgrastim was about 50 hours. The elimination half-life is
reported to be 3-4 hours for filgrastim and 42-67.5 hours (depending on dose) for
pegfilgrastim.

e, Immunogenicity
[0380] Among the study participants, there was one confirmed anti-G-CSF/neoepitope
antibody response in the Neugranin-treated subjects and one anti-G-CSF response in the
pegfilgrastim-treated group, or 0.5% and 0,9%, respectively (Table 30). In both cases, the
subjects had elevated non-specific binding in pre-dose samples.

[0381] After NEUG treatment, very low levels of confirmed positive antibodies were seen
in the patient, with no apparent increase in the magnitude of the response after repeated doses
(data not shown). In the pegfilgrastim-treated patient, an unusually high non-specific
background binding was observed; however, only a transient confirmed antibody response
was seen after Cycle 2 treatment (data not shown). No antibody response was neutralizing.
[0382] Anti-HSA antibodies were naturally occurring at a low level in this population, with
6.9% of the subjects testing positive for HSA antibodies in the pre-dose evaluation.
Treatment emergent anti-HSA antibodies were observed in four NEUG-treated subjects,
1.8% (Table 31). All responses were transient and weak. Three responses emerged after the
first treatment cycle and were undetectable after Cycles 2,3 and 4. One response occurred
after the third treatment but was undetectable at the 30 day follow-up after the 4th treatment
(data not shown).



f. Treatment-emergent adverse events in Phase II, Part B
[0383 J In Phase II, Part B, >90% of subjects in each treatment group experienced at least
one treatment-emergent adverse event (TEAE), and the percent of subjects with at least one
TEAE related to study medication ranged from 23.1% in the pegfilgrastim group to 35.0% in
the Neugranin 50 mg group. The percent of subjects with at least one SAE was highest in the
NEUG 30 mg group (30%), but was approximately 15% in the other three treatment groups.
None of the SAEs were related to study medication. One patient (NEUG 30 mg) was
withdrawn from the study due to diabetic foot, which was considered to be not related to
study medication. In the Part B, ail except 8 subjects (2 NEUG 40 mg, 3 NEUG 50 mg, 3
pegfilgrastim) had at least one TEAE. The percent of subjects with at least one TEAE related
to study medication was 20.2% in the NEUG 50 mg group, 22.4% in the NEUG 40 mg group
and 22.1% in subjects receiving pegfilgrastim. Two subjects (1 NEUG 40 mg, 1
pegfilgrastim) died during the study, and 6-8 subjects in each treatment group experienced
at least one SAE. No deaths or SAEs were considered to be related to study medication.
[0384] The total number of TEAEs was similar across treatment groups in Part A, when
sample size is taken into consideration for the NEUG 30 mg dose, and in Part B. In both
Parts A and B, the percent of TEAEs with CTC Grade 3 or higher was similar for NEUG and
pegfilgrastim as was the percent of TEAEs related to study medication.
g. Dose response
[0385] The results of Phase II demonstrated that both 40 and 50 mg fixed doses of NEUG
provided equivalent safety and efficacy to 6 mg of pegfilgrastim in breast cancer subjects
treated with myelotoxic chemotherapy. While the mean DSN for the 40 mg treatment group
was slightly lower than the mean DSN of the 50 mg group, these differences were not
statistically significant. A dose response was observed for AUCANC (Days 0-15 in cycle 1)
both when weight-adjusted dose was considered and for fixed dose cohorts (FIG. 24). The

AUCANC for the 30 mg cohort was slightly lower than that of pegfilgrastim, indicating that
the 30 mg fixed dose was less effective in this study, whereas AUCANC for the 40 mg and the
50 mg cohorts were dose-related and higher (although not significantly) than the AUCANC for
pegfilgrastim treated subjects. From the above analysis, a dose response is apparent when
NEUG is administered on a weight adjusted basis (mg/kg). However, comparison of DSN in
cycle 1 for Phase II, Part B suggested that patients of all weight quartiles were adequately
supported as DSN did not vary significantly among the treatment arms (40 and 50 mg NEUG
and pegfilgrastim) nor with weight-adjusted dose (mg/kg). Further, there was no evidence
that a fixed dose might result in an altered safety profile in lighter patients as the incidence
and severity of related adverse events (bone pain in particular; data not shown) did not
correlate with dose received per kilogram body weight, nor were they different from those
with pegfilgrastim.
Example 12: Additional exemplary NEUG formulations
[0386] Development of additional NEUG formulations was undertaken to analyze and
determine the effects of pH (4-7), buffer species (L-histidine, citrate, acetate), tonicifiers
(sorbitol vs. NaCl), and protein concentration (concentrated up to 70-90 mg/ml). Samples
were analyzed/monitored via SEC (monomer %), RH-HPLC (purity %), Eliman's assay (-SH
amount), A280 (protein), and IE-HPLC (charge isoforms). The formulations examined are
presented in Table 32, below.
[0387] After testing, formulations A40S, A45S, C55N and C55S appeared the most stable
(in explored aspects).



[0388] It will be apparent to those skilled in the art that various modifications and
variations can be made in the methods and compositions of the present invention without
departing from the spirit or scope of the invention. Thus, it is intended that the present
invention cover the modifications and variations of this invention provided they come within
the scope of the appended claims and their equivalents.

WHAT IS CLAIMED IS:
1. A pharmaceutical composition comprising recombinant human albumin-human
granulocyte colony stimulating factor and at least one pharmaceutically acceptable
carrier, wherein the composition has a pH of between about 4 and about 6.8.
2. The pharmaceutical composition of claim 1 wherein the composition has a pH of about
6.0.
3. The pharmaceutical composition of claim 1, wherein the concentration of recombinant
human albumin-human granulocyte colony stimulating factor is between about 2.5 and
about 240 mg/ml.
4. The pharmaceutical composition of claim 1, wherein the concentration of recombinant
human albumin-human granulocyte colony stimulating factor is between about 30 and
about 120 mg/ml.
5. The pharmaceutical composition of claim 1, wherein the concentration of recombinant
human albumin-human granulocyte colony stimulating factor is between about 60 and
about 120 mg/ml.
6. The pharmaceutical composition of any one of claims 1-5 comprising at least one
pharmaceutically acceptable salt.
7. The pharmaceutical composition of claim 6 wherein the concentration of salt is between
about 5 and about 50 mM.
8. The pharmaceutical composition of any one of claims 1 -7, comprising a pharmaceutically
acceptable buffer.
9. The pharmaceutical composition of claim 8 wherein the concentration of buffer is
between about 15 and about 50mM.
10. The pharmaceutical composition of claim 8 wherein the concentration of buffer is about
20mM.

11. The pharmaceutical composition of any one of claims 8-10, wherein the buffer is a
phosphate or a citrate.
12. The pharmaceutical composition of any one of claims 8-11, wherein the buffer is sodium
phosphate.
13. The pharmaceutical composition of claim 12 wherein the buffer comprises sodium
phosphate monobasic or sodium phosphate dibasic.
14. The pharmaceutical composition of any one of claims 1-13, comprising a freeze-drying
stabilizer.
15. The pharmaceutical composition of claim 14 wherein the freeze-drying stabilizer
comprises trehalose dihydrate.
16. The pharmaceutical composition of claim 15 wherein the concentration of trehalose
dihydrate is about 60mM.
17. The pharmaceutical composition of any one of claims 1-16, comprising a bulking agent.
18. The pharmaceutical composition of claim 17 wherein the bulking agent comprises a poly-
alcohol.
19. The pharmaceutical composition of claim 18 wherein the poly-alcohol comprises
mannitol.
20. The pharmaceutical composition any one of claims 1-19, in the form of a lyophilized
powder.
21. The pharmaceutical composition of claim 20 wherein the powder is stored in a vial.
22. The pharmaceutical composition of any one of claims 1-19, wherein the composition is in
the form of a liquid.
23. The pharmaceutical composition of claim 22, wherein the liquid is stored in a syringe.
24. The pharmaceutical composition according to any one of claims 1-23, comprising:
(a) recombinant human albumin-human granulocyte colony stimulating factor,

and
(b) at least one buffer;
wherein the monomeric purity in solution of the recombinant human albumin-human
granulocyte colony stimulating factor decreases by less than 10% after incubation at 25°C
for 24 hours.
25. The pharmaceutical composition according claim 1, comprising:
(a) recombinant human albumin-human granulocyte colony stimulating factor,
(b) 20 mM sodium phosphate,
(c) 180 mmmannitol,
(d) 60 mM trehalose dihydrate,
(e) 0.01% (W/V) polysorbate 80,
wherein the composition has a pH of about 6.0 and wherein the concentration of the
recombinant fusion protein is between 2.5 and 120 mg/ml.
26. The pharmaceutical composition according to claim 1, comprising recombinant human
albumin-human granulocyte colony stimulating factor and PMTT20/6.0, wherein the
concentration of the recombinant fusion protein is between about 2.5 and about 120
mg/ml.

Described herein are compositions and methods for treating, preventing and ameliorating diseases and conditions
characterized by a lower than normal white blood cell count, such as leukopenia and neutropenia. The compositions and methods
include recombinant human albumin-human granulocyte colony stimulating factor. Pharmaceutical formulations including the recombinant
fusion protein, and methods of making such formulations are also described.